Emtricitabine [(-)FTC; (-)-beta-L-2'-3'-dideoxy-5-fluoro-3'-thiacytidine] is an oxathiolane nucleoside analog recently approved by the Food and Drug Administration for the treatment of human immunodeficiency virus (HIV). Structurally, (-)FTC closely resembles lamivudine [(-)3TC] except that the former is 5-fluorinated on the cytosine ring. In HIV-1 reverse transcriptase (RT) enzymatic assays, the triphosphate of (-)FTC [(-)FTC-TP] was incorporated into both DNA-DNA and DNA-RNA primer-templates nearly 3- and 10-fold more efficiently than (-)3TC-TP. Animal studies and clinical trial studies have demonstrated a favorable safety profile for (-)FTC. However, a detailed study of the incorporation of (-)FTC-TP by human mitochondrial DNA polymerase gamma, a host enzyme associated with nucleoside toxicity, is required for complete understanding of the molecular mechanisms of inhibition and toxicity. We studied the incorporation of (-)FTC-TP and its enantiomer (+)FTC-TP into a DNA-DNA primer-template by recombinant human mitochondrial DNA polymerase in a pre-steady-state kinetic analysis. (-)FTC-TP was incorporated 2.9 x 10(5)-, 1.1 x 10(5)-, 1.6 x 10(3)-, 7.9 x 10(3)-, and 100-fold less efficiently than dCTP, ddCTP, (+)3TC-TP, (+)FTC-TP, and (-)3TC-TP, respectively. The rate of removal of (-)FTC-MP from the corresponding chain-terminated 24-mer DNA by polymerase gamma's 3'-->5' exonuclease activity was equal to the removal of (+)FTC-MP, 2-fold slower than the removal of (-)3TC-MP and (+)3TC-MP, and 4.6-fold slower than the excision of dCMP. These results demonstrate that there are clear differences between HIV-1 RT and polymerase gamma in terms of preferences for substrate structure.