"The Islamic emirate (of Afghanistan) once again informs all the people that no one should participate in this American process and should boycott the process," said a Taliban statement emailed to AFP.
"The mujahedeen are fully prepared to defeat this process," it said, adding: "Anyone who participates and gets hurt will be responsible for their own losses."
The run-off sees former foreign minister Abdullah Abdullah take on the incumbent Hamid Karzai, who came to power in late 2001 after the Taliban were toppled by US-led forces.
Eight years on, the Islamists are waging an increasingly virulent insurgency, particularly in their southern heartland, where attacks and threats helped limit turnout in some provinces such as Kandahar to ten percent or less.
Almost 200 violent incidents around the first vote were attributed to the Taliban, including amputations of fingers marked with purple ink as proof of voting, and rocket and grenade attacks on polling stations.
Tribal chiefs from across the war-torn country met in Kabul on Saturday to discuss security.
"This time it's believed that more people will participate in the election," said Mohammad Nasim, an elder from southern Logar province.
Almost one million of Karzai's share of the preliminary results -- around one-third of all votes cast for him -- were eliminated for fraud, cutting his lead to below the 50 percent threshold needed for outright victory.
Karzai told CNN that British Prime Minister Gordon Brown and other world leaders personally pressured him to accept a second round, which he did out of security concerns and commitment to "democratic traditions."
"There was this friendly effort by some governments to ask me to accept the result of the ECC (Electoral Complaints Commission)," which is backed by the United Nations, he said in an interview to be broadcast in full Sunday.
Speculation persists that Karzai and Abdullah could reach a deal that will negate the need for the run-off -- perhaps through a power-sharing deal or with Abdullah bowing out of the second presidential race.
But in an interview with CNN, Abdullah ruled out joining Karzai's government should he lose the run-off.
"I think I left Mr. Karzai's government some three-and-a-half years ago, and since then I've not been tempted to be part of that government... part of the same deteriorating situation," he said.
Karzai's campaign spokesman Waheed Omar also dismissed any power-sharing deal.
"For us the only constitutional way of establishing a new government is to go to a second round," he said.
Abdullah, who travelled to most of Afghanistan's 34 provinces during first-round campaigning, will mainly stay in Kabul this time, his spokesman Sayed Aqa Fazil Sancharaki said.
The run-off has been proclaimed as a chance to revive Afghanistan's democratic ambitions and bring some credibility to the election process, supported and bankrolled by the UN to the tune of 380 million dollars.
The UN's envoy to Kabul, Kai Eide, also said he expects less ballot-stuffing than in the first round, though he gave no details of how this will be achieved other than by sacking some 200 officials implicated in the August 20 fraud.
Richard Holbrooke, US special envoy to Afghanistan, told reporters he believed a second round would be less troubled by fraud or security concerns.
"One, there are only two candidates. Two, there's the experience factor. Three, the international community, including the forces under General (Stanley) McChrystal's command, are going to go all out to help make this a success."
He added that McChrystal will have more troops to deploy than he had for the first round.
McChrystal has requested an extra 40,000 troops to add to the more than 100,000 already in Afghanistan fighting the Taliban under US and NATO command.
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