Zidovudine (AZT) is a reverse transcriptase (RT) inhibitor widely used to treat persons infected with HIV-1 and HIV-2. Recent data on treated patients suggest differences in the antiviral activity of AZT between HIV-1 and HIV-2. We evaluated the antiviral activity of AZT on HIV-2 by using multiple approaches including in vitro selection experiments, analysis of growth kinetics with AZT, and phenotypic testing. A total of 5 wild-type (WT) HIV-2 viruses were used in the analysis. For comparison, 4 control WT HIV-1 strains and one HIV-1 mutant carrying the 215S mutation were evaluated in parallel. All 5 HIV-1 isolates acquired AZT resistance mutations after 3-6 passages with AZT or a 4- to 32-fold increase in AZT concentration. Among these viruses, the fastest selection of resistance was seen in HIV-1(S215), which acquired S215Y (1-nucleotide change only) at passage 3 after only 17 days in culture. In contrast, none of the 5 HIV-2 viruses that naturally have S215 acquired S215Y/F or any other RT mutation during 10 passages with AZT (1025-fold increase in AZT concentration). In the presence of AZT + didanosine (ddI), 3 of the 5 HIV-1 isolates acquired AZT or ddI resistance mutations, while only ddI resistance mutations were seen in HIV-2 (4 of 5 isolates). All HIV-2 viruses replicated efficiently in high AZT concentrations and were about 200-fold less sensitive to AZT than HIV-1. In contrast, HIV-2 and HIV-1 were equally susceptible to ddI, a finding consistent with the selection of HIV-2 mutants with AZT + ddI. Our results demonstrate that the activity of AZT on HIV-2 is lower than previously thought, and emphasize the need for novel antiretroviral drugs specific for HIV-2.