Optimal management of human immunodeficiency virus type 1 (HIV-1) infections may require combinations of anti-HIV-1 agents. Zidovudine (AZT, 3'-azido-3'-deoxythymidine), didanosine (ddI, 2',3'-dideoxyinosine), and recombinant interferon-alpha A (rIFN-alpha A) were evaluated in two-drug regimens against replication of AZT-resistant HIV-1 in vitro. AZT-sensitive and AZT-resistant isolate pairs derived from two individuals before and after extended AZT monotherapy were studied. Drug interactions using peripheral blood mononuclear cells infected with HIV-1 were evaluated mathematically. Synergistic interactions were seen among AZT, ddI, and rIFN-alpha A in two-drug regimens against AZT-resistant HIV-1 in vitro, even when AZT was included in the treatment regimen. Mixtures of wild-type and mutant reverse transcriptase genes were found in one of the late-AZT therapy isolates, suggesting that the mechanism of synergy of AZT-containing regimens may involve inhibition of AZT-sensitive viruses in the viral pool. These studies suggest that AZT may be useful in drug combination regimens, even when AZT-resistant viruses are isolated in vitro.