Enantiomers of beta-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine (D/L-D4FC) are nucleoside analog reverse transcriptase inhibitors (NRTIs) currently under investigation as antiviral agents. One of the major problems of NRTIs is toxicity to mitochondria. It has been shown that mitochondrial toxicity of NRTIs can correlate with incorporation and removal of these compounds by mitochondrial DNA polymerase (Pol gamma). Mechanistic studies have shown that, if activated, NRTIs are incorporated more efficiently by HIV-1 reverse transcriptase (RT) and less efficiently by Pol gamma, the corresponding nucleosides are considered to be more selective. In the present study, in order to predict potential DNA Pol gamma-related mitochondrial toxicity of D- and L-D4FC, the incorporation and removal of the monophosphate form of these compounds by Pol gamma were studied using transient kinetic methods. Our cell-free results showed that Pol gamma incorporated the natural D-isomer significantly more efficiently than the unnatural L-isomer. However, the removal rates of these enantiomers from the chain-terminated primers were almost identical. While these results suggest that D-D4FC may present more mitochondrial toxicity than L-D4FC in cell-free assays, we have previously shown that HIV-1 RT prefers D-D4FC-TP as a substrate over the L-isomer, particularly in the case of mutant forms of RT associated with nucleoside drug resistance such as M184V. Since the effectiveness of NRTIs is a balance between efficiency of incorporation by wild-type and drug-resistant forms of HIV-1 RT and mitochondrial toxicity, our kinetic results suggest that both enantiomers may show promise as potential therapeutics.