The triphosphorylated form of the nucleoside analogue AZT (AZTTP) acts as a chain terminator during reverse transcription of the human immunodeficiency virus (HIV) genome. The bottleneck in the conversion of AZT to AZTTP is the phosphorylation of AZT monophosphate (AZTMP) by cellular thymidylate kinase. Human thymidylate kinase was engineered to exhibit highly improved activity for AZTMP to AZTDP conversion. It was demonstrated here that genetically modified human cells transiently expressing these enzyme variants show more than 10-fold higher intracellular concentrations of AZTDP and AZTTP. Stable clones expressing these enzymes appear to phosphorylate AZTMP less efficiently, but first experiments indicate they are still more potent in HIV inhibition than the parental cells. It was proposed that the concept of introducing into human cells a catalytically improved human enzyme, rather than an enzyme of viral, bacterial or yeast origin, may serve as a paradigm for ameliorating the metabolic activation of an established drug.