Among the acquired immunodeficiency syndrome (AIDS) drugs approved by the FDA for clinical use, two are modified cytosine analogs, Zalcitabine (ddC) and Lamivudine [(-)3TC]. (-)3TC is the only analog containing an unnatural L (-) nucleoside configuration. Similar to other dideoxy nucleosides, these analogs are metabolically activated to the triphosphate that is incorporated into DNA by human immunodeficiency virus type 1 (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 2',3'-dideoxycytidine (ddC). The mechanistic basis for the stereochemical selectivity and differential toxicity of the isomeric 2',3'-dideoxy-3'-thiacytidine (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 analogs by comparing the effects of 2',3'-deoxycytidine-5'-triphosphate (ddCTP), beta-D-(+)-2'3'-dideoxy-3'-thiacytidine-5'-triphosphate [(+)3TC-TP] and beta-L-(-)-2'3'-dideoxy-3'-thiacytidine-5'-triphosphate [(-)3TC-TP] on the HIV-1 RT as well as a recombinant form of the human mitochondrial DNA polymerase gamma (Pol gamma), the holoenzyme polymerase responsible for mitochondrial DNA replication. In this review, we discuss studies which may provide insight into the molecular mechanism for the stereochemical selectivity and differential toxicity.