Mitochondrial toxicity is a limiting factor in the use of some nucleoside reverse transcriptase inhibitors of HIV. To further understand the impact of structural features on the incorporation and exonuclease removal of nucleoside monophosphate (MP) analogues by human mitochondrial DNA polymerase (pol gamma), transient kinetic studies were done with analogues of 2'-deoxyguanosine triphosphate. The kinetic parameters for the incorporation and removal of carbovir (CBV)-MP, dioxolane guanosine (DXG)-MP and 2',3'-dideoxy-2',3'-didehydroguanosine (d4G)-MP were studied with pol gamma holoenzyme. The importance of the ribose oxygen in incorporation by pol gamma was illustrated by an approximate 3,000-fold decrease in the incorporation efficiency of an analogue lacking the ribose oxygen (CBV-TP) relative to those containing a ribose oxygen (DXG-TP and d4G-TP). As a result, a comparison with previous data for the incorporation by HIV reverse transcriptase showed CBV-TP to be approximately 800-8,000-fold more selective for its antiviral target over pol gamma relative to the other guanosine analogues. However, DXG-TP and d4G-TP were found to be much more selective than previously reported values for mitochondrial toxic nucleoside analogues. Structural modelling based on sequence homology with other polymerase A family members suggests that an interaction between the ribose oxygen and arginine 853 in pol gamma may play a critical role in causing this differential incorporation. Exonuclease removal of a chain-terminating CBV-MP was also found to be more efficient by pol gamma. These results help to further elucidate the structure activity relationships for pol gamma and should aid in the design of more selective antiviral agents.