Background: Entecavir, a guanosine nucleoside analog, was thought to have selective activity against the Hepatitis B virus (HBV) polymerase. However, we recently reported that entecavir is a potent partial inhibitor of HIV reverse transcriptase (RT) and that in some patients, it selects HIV variants resistant to the important anti-HIV drugs lamivudine and emtricitabine. We have done additional studies to understand the evolution of drug resistant HIV in patients taking entecavir monotherapy. Methods: For the phylogenetic analysis of HIV variants, we used RT-PCR and cloning to sequence independent viruses from patient plasma. In vitro studies were performed using a single round HIV infectivity assay. Results: Extensive phylogenetic analysis of HIV variants in HBV-HIV co-infected patients on entecavir monotherapy documented the emergence of the M184V mutation in HIV RT. In vitro studies demonstrate a complex relationship between the fitness cost and fitness advantage of the M184V mutation relative to wild type virus in the absence and presence of relevant in vivo entecavir concentrations. In some cases, the measured replication of mutant virus in the presence of entecavir is only slightly higher than the replication of the wild type virus in the presence of ETV, reflecting the substantial fitness cost of the mutation and the modest inhibitory activity of the drug. Additional in vitro experiments implicate cellular factors which influence the selective pressure exerted by entecavir in vivo. Conclusions: These data provide an explanation for the variable rate at which M184V variants emerge in patients on entecavir monotherapy. The results highlight the clinical significance of the anti-HIV activity by entecavir and provide a basis for understanding the evolution of drug resistant HIV in patients taking entecavir.