An azidothymidine (AZT)-resistant virus strain (HIV-1/AZT) (containing the 67 Asp --> Asn, 70 Lys --> Arg, 215 Thr --> Phe and 219 Lys --> Gln mutations into its reverse transcriptase) was grown in the combined presence of 2',3'-dideoxy-3'-thiacytidine (3TC, lamivudine) and the nonnucleoside reverse transcriptase inhibitor (S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3,4-dih ydroquinoxaine-2(1H)-thione (quinoxaline HBY 097). Replication of HIV-1/AZT was inhibited to a significantly greater extent by the combination of 3TC and quinoxaline HBY 097 than by either drug alone. Virus breakthrough was markedly delayed in the combined presence of 3TC and HBY 097 at drug concentrations as low as 0.05 microg/mL and 0.0025 microg/mL, respectively. The virus that was recovered after exposure to the compounds (3TC and HBY 097) individually had acquired, in the genetic AZT-resistance background of HIV-1/AZT, 103 Lys --> Glu and 106 Val --> Ala mutations. The 103 Lys --> Glu mutation had not been observed before. However, both virus mutants retained marked sensitivity to HBY 097. In all cases, the genotypic AZT-resistance mutations were maintained in the mutant virus RT genomes, and the viruses also remained phenotypically resistant to AZT. Given the exquisite potency of a concomitant combination of 3TC and HBY 097 in suppressing virus replication, this drug combination should be further pursued in clinical trials in HIV-1-infected individuals.