The antiviral activity against human immunodeficiency virus type 1 of the two structurally related thymidine analogs azidothymidine and fluorothymidine, both alone and in combination, was tested. Fluorothymidine was tenfold more active than azidothymidine. The selectivity indices of the two compounds were similar. The combination of azidothymidine and fluorothymidine showed clearly synergistic antiviral activity, and diminished cytotoxicity. The inhibition of reverse transcriptase from human immunodeficiency virus type 1 by the triphosphates of azidothymidine and fluorothymidine, both alone and in combination was also tested. Azidothymidine triphosphate was a fourfold stronger inhibitor than fluorothymidine triphosphate. The combination of the two showed only additive (and not synergistic) effects upon reverse transcriptase. The combination of azidothymidine and fluorothymidine showed both synergistic antiviral activity and diminished cytotoxicity, and may therefore represent a promising therapeutic strategy. The additive (and not synergistic) inhibition of reverse transcriptase by the combination of the triphosphates indicates that in cell culture additional factors other than inhibition of the reverse transcriptase by the triphosphates influence the antiviral activity of the combination. Such factors might include effects upon normal nucleoside metabolism or metabolism of the analogs. Alternatively, one of the nucleosides might have an additional mechanism of action besides inhibition of the reverse transcriptase.