To examine the role of the mitochondrial polymerase (Pol g) in clinically observed toxicity of nucleoside analogs used to treat AIDS, we examined the kinetics of incorporation catalyzed by Pol g for each FDA-approved analog plus FIAU, (-)3TC and PMPA. We used recombinant exonuclease-deficient (E200A), reconstituted human Pol g holoenzyme in single turnover kinetic studies to measure Kd (Km) and kpol (kcat) to estimate the specificity constant (kcat/Km) for each nucleoside-analog triphosphate. The specificity constants vary over 500,000-fold for the series: ddC > FIAU > ddA (ddI) > d4T >> (+)3TC >> (-)3TC > PMPA > AZT >> CBV. Abacavir (prodrug of CBV) and PMPA are two new drugs that are expected to be least toxic. Notably, the higher toxicities of d4T, ddC and ddA arose from their 13 to 36-fold tighter binding relative to the normal dNTP even though their rates of incorporation were comparable to PMPApp and AZT. We also examined the rate of exonuclease removal of each analog following incorporation. The rates varied from 0.06 to 0.0004 s-1 for the series: FIAU > (+)3TC ~ (-)3TC > CBV > AZT > PMPA ~ d4T >> ddA (ddI) >> ddC. Removal of ddC was too slow to measure (<0.00002 s-1). The high toxicity of dideoxy compounds, ddC and ddI (metabolized to ddA) may be a combination of high rates of incorporation and ineffective exonuclease removal. Conversely, the more effective excision of (-)3TC, CBV and AZT may contribute to lower toxicity. FIAU is readily extended by the next correct base pair (0.13 s-1) faster than it is removed (0.06 s-1) and therefore is stably incorporated and highly mutagenic. We define a toxicity index for chain terminators to account for relative rates of incorporation versus removal. These results provide a method to rapidly screen new analogs for potential toxicity.