Lamivudine therapy selects for the M184V mutation. Although this mutation reduces the replicative capacity of human immunodeficiency virus in vitro, its impact on viral fitness in vivo has not been well defined. We used quantitative allele-specific PCR to precisely calculate the fitness differences between the mutated M184V virus and one that had reverted to the wild type in a cohort of patients by selectively interrupting reverse transcriptase inhibitor therapy, and we found that the M184V variants were consistently 4 to 8% less fit than the wild type in the absence of drug. After a lag phase of variable duration, wild-type variants emerged due to continued evolution of pol and back mutation rather than through emergence of an archived wild-type variant. Copyright © 2009, American Society for Microbiology. All Rights Reserved.