Two cis stereoisomers of 2',3'-dideoxy-3'-thiacytidine (SddC) were investigated for their activity against human immunodeficiency virus type 1 (HIV-1) in human acute lymphoblastic leukemia H-9 cells. (-)-SddC is six times more potent against HIV-1 and at least 1.7-fold less cytotoxic than (+)-SddC. Metabolism studies showed that the intracellular accumulation of the active triphosphate form of (-)-SddC is more than 2-fold greater than that of (+)-SddCTP in H-9 cells. In contrast, (+)-SddCTP is approximately 1.5 times more potent than (-)-SddCTP as an inhibitor of HIV-1 reverse transcriptase using a rRNA template (Ki = 0.22 and 0.034 microM, respectively) and gapped DNA (Ki = 0.53 and 1.02 microM, respectively). The enantiomers are comparable as substrates for incorporation into DNA by the RNA-dependent HIV-1 reverse transcriptase; however, neither analog is incorporated as readily as dCTP. The above observations do not explain the difference in the anti-HIV potency between the enantiomers. A novel 3'-5' exonuclease was partially purified from the cytosol of H-9 cells and assayed for the removal of (+)- and (-)-SddCMP-terminated DNA. Removal of (+)-SddCMP was approximately two to three times faster from 3'-terminals of single-stranded and double-stranded DNA, whereas on DNA/RNA substrates, the exonucleolytic cleavage of (+)-SddCMP proceeded approximately six times faster than that of (-)-SddCMP. This result correlates with the observed difference in the anti-HIV effect between the two compounds and suggests that this novel enzyme may be an important determinant of their antiviral activities.