A novel non-chain terminating nucleoside analog anti-HIV inhibitor, KP-1212 has been designed to form base pairs with multiple bases that may lead to mutagenesis in the HIV-1 viral genome. After multiple replication cycles, the accumulation of mutations surpasses a crucial threshold beyond which the virus can no longer replicate. HIV-1 reverse transcriptase (RT) incorporates the KP-1212 monophosphate into the genome during viral replication after metabolic activation of the KP-1212 nucleoside to the triphosphate. The propensity for forming alternate base pairs with the KP-1212 nucleotide leads to mismatched nucleotides and the subsequent misincorporation is the basis for the inhibitory activity. The results showed that HIV-1 RT and human mitochondrial DNA polymerase (Pol gamma) incorporated KP-1212-TP with a significant level of efficiency, whereas mouse DNA polymerase beta (Pol beta) did not. Misincorporation studies suggest that both HIV-1 RT and Pol gamma may cause mutations at significantly high rates. These in vitro data confirm the mechanistic basis of KP-1212 as a viral mutagen but suggest that there may be a potential for toxicity to the mitochondria.