Spontaneous mutation rates at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus were measured in human cancer cell lines defective in the mismatch repair (MMR) genes hMLH1, hPMS2, or GTBP, as well as in a cell line carrying mutations in both hMLH1 and hPMS2. The mutation rate was determined by quantitating mutant frequency increases within a single culture as a function of cell division. These MMR-deficient cell lines exhibited a 50- to 750-fold increase in mutation rate relative to a MMR-proficient cancer cell line. From lowest to highest, the spontaneous mutation rates relative to the MMR-gene defects studied here are as follows: hMLH1- < GTBP- < hPMS2- < hMLH1- / hPMS2-. In addition, a cell line in which MMR was restored by chromosome transfer exhibited a mutation rate 12-fold below the MMR-deficient parental cell line. These data support the notion that MMR plays an important role in controlling the rate of spontaneous mutation and suggest that different MMR-gene defects may vary in their ability to repair different types of DNA mismatches, thus leading to measurable quantitative differences in spontaneous mutagenesis. Furthermore, a difference in mutation rates was observed between a hPMS2-defective cell line (3.1 x 10(-5) mutations/cell/generation) and two hMLH1-defective cell lines (4.0 x 10(-6) and 7.3 x 10(-6) mutations/cell/generation). Assuming the hPMS2- and hMLH1-gene products only function in the proposed hMutL alpha heterodimer, then defects in either gene should yield comparable mutation rates. These data suggest that hPMS2 plays a critical role in MMR, while additional hMLH1 homologues or hPMS2 alone may function to partially complement defects in hMLH1.