Among the AIDS drugs approved by the US Food and Drug Administration (FDA) for clinical use, two are modified cytosine analogues, zalcitabine (ddC) and lamivudine [(-)3TC]. (-)3TC is the only analogue containing an unnatural L(-)nucleoside configuration. Similar to other dideoxynucleosides, these analogues are metabolically activated to the triphosphate that is incorporated into DNA by HIV-1 reverse transcriptase (RT), resulting in DNA chain termination and ultimately cessation of viral replication. The natural d(+)3TC isomer also acts in a similar manner to inhibit HIV-1 RT. In cell culture (-)3TC is less toxic than its d(+)isomer (+)3TC, containing the natural nucleoside configuration, and both are considerably less toxic than ddC. The mechanistic basis for the stereochemical selectivity and differential toxicity of the isomeric 3TC and ddC compounds is not completely understood, although a number of factors may clearly come into play. We have previously investigated the mechanistic basis for the differential stereoselective inhibition and toxicity of these three cytosine analogues by comparing the effects of ddCTP (+)3TC-TP and (-)3TC-TP on the HIV-1 RT, as well as a recombinant form of the human mitochondrial DNA polymerase (Polgamma), the holoenzyme polymerase responsible for mitochondrial DNA replication. In this paper, we discuss the molecular mechanism for the stereochemical selectivity and differential toxicity.