(-)-2'-Deoxy-3'-thiacytidine (3TC) is a selective inhibitor of human immunodeficiency virus replication in vitro (J. A. V. Coates, N. Cammack, H. J. Jenkinson, A. J. Jowett, M. I. Jowett, B. A. Pearson, C. R. Penn, P. L. Rouse, K. C. Viner, and J. M. Cameron, Antimicrob. Agents Chemother. 36:733-739, 1992). The effect of 3TC 5'-triphosphate on both the RNA-dependent and DNA-dependent activities of human immunodeficiency virus type 1 reverse transcriptase and DNA polymerases alpha, beta, and gamma from HeLa cells was investigated. 3TC 5'-triphosphate is a competitive inhibitor (with respect to dCTP) of the RNA-dependent DNA polymerase activity (apparent Ki = 10.6 +/- 1.0 to 1.24 +/- 5.1 microM, depending on the template and primer used); the DNA-dependent DNA polymerase activity is 50% inhibited by a 3TC 5'-triphosphate concentration of 23.4 +/- 2.5 microM when dCTP is present at a concentration equal to its Km value. Chain elongation studies show that 3TC 5'-triphosphate is incorporated into newly synthesized DNA and that transcription is terminated in a manner identical to that found for ddCTP. The 50% inhibitory concentrations of 3TC 5'-triphosphate against DNA polymerases alpha, beta, and gamma at concentrations of dCTP equal to the Km were 175 +/- 31, 24.8 +/- 10.9, and 43.8 +/- 16.4 microM, respectively. More detailed kinetic studies with 3TC 5'-triphosphate and DNA polymerases beta and gamma are consistent with the fact that inhibition of these enzymes by 3TC 5'-triphosphate is competitive with respect to dCTP. The values of Ki were determined to be 18.7 microM for DNA polymerase beta and 15.8 +/- 0.8 microM for DNA polymerase gamma.