Mayo is a former Dauphine Libere race winner
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Spain's Iban Mayo was given a two-year ban after the Court of Arbitration for Sport (Cas) upheld an appeal by the International Cycling Union (UCI).
The UCI took its case to Cas after the Spanish Cycling Federation refused to punish the rider.
The 30-year-old, who has not raced this season, tested positive for EPO during the 2007 Tour de France, but challenged the legitimacy of the B test.
Cas ruled the ban should be upheld after a second B test proved positive.
In a statement on Tuesday, the court said: "The Cas panel was of the opinion that the decision of the UCI to proceed to a new analysis of the B sample was in accordance with the letter and spirit of the UCI Anti-Doping Regulations."
Mayo's suspension is backdated to start from 31 July 2007, the day on which he was sacked by his team Saunier Duval.
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606: DEBATE
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The initial B sample was returned by a different laboratory than the one which carried out the initial test, causing Mayo to question its legitimacy.
The UCI instructed the original laboratory to do its own B test and called for the rider to be banned after that came back positive.
The mountain specialist, who finished the 2007 Tour in 16th place overall but was later disqualified, has said he is considering taking legal action against the UCI over the way he has been treated.
"It was a very bad experience because I didn't understand what was happening but everything has turned out as I expected," he told Spanish newspaper Marca.
"It doesn't seem logical nor credible," he said. "I've spent many years cycling and I can't chuck it all in but sometimes you feel like it because there are so many injustices.
"The fans who like cycling don't want to see this sort of thing. Cycling is a spectacle that involves sacrifice - if not you don't get the results."
Mayo had been tipped to shine in the Tour de France but failed to live up to expectations.
He finished sixth in the Tour in 2003 and won the Dauphine Libere race in 2004.
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