Mitochondrial mutational spectra in human cells, tissues and derived tumors for bp 10,030-10,130 are essentially identical, suggesting a predominant mutagenic role for endogenous processes. We hypothesized that errors mediated by mitochondrial DNA polymerase gamma were the primary sources of mutations. Point mutations created in this sequence by human DNA pol gamma in vitro were thus compared to the eighteen mutational hotspots, all single base substitutions, previously found in human tissues. The set of concordant hotspots accounted for 83% of these in vivo mutational events. About half of these mutations are insensitive to prolonged heating of DNA during PCR and half increase proportionally with heating time at 98 degrees C. Primary misincorporation errors and miscopying errors past thermal denaturing products such as deaminated cytosines (uracils) thus appear to be of approximately equal importance. For the sequence studied, these data support the conclusion that, endogenous error mediated by DNA pol gamma constitutes the primary source of mitochondrial point mutations in human tissues.