We evaluated the effect of drug alternation on human immunodeficiency virus type 1 (HIV-1) suppression and emergence of resistant virus strains in CEM cell cultures. Rapid, that is, twice weekly, alternation of lamivudine and nevirapine (at 0.1 microg/ml) at the time of each subcultivation resulted in a long-term suppression of virus replication (up to 18 subcultivations, or 9 weeks) and eventual sterilization of the cell cultures. Under similar experimental conditions mutant virus rapidly emerged (within two or three subcultivations) when the same drug (lamivudine or nevirapine) was applied throughout. The longer the HIV-1-infected cell cultures were exposed to the same drug before shifting to the other drug, the shorter the time period for mutant virus to appear. Those virus strains that eventually emerged under suboptimal alternating drug therapy harbored a combination of lamivudine- and nonnucleoside reverse transcriptase (RT)-specific mutations in the RT gene. When extrapolated to the clinical setting, our data indicate that the efficacy of HIV-1 therapy may be markedly improved by rapid alternation of drug cocktails before detectable evidence of resistance development as compared with drug cocktail alternation after the first signs of drug resistance become visible.