DNA polymerase beta functions in both base excision repair and meiosis. Errors committed by polymerase beta during these processes could result in mutations. Using a complementation system, in which rat DNA polymerase beta substitutes for DNA polymerase I of Escherichia coli, we previously isolated a DNA polymerase beta mutant in which Tyr-265 was altered to Cys (Y265C). The Y265C mutant is dominant to wild-type DNA polymerase beta and possesses an intrinsic mutator activity. We now have expressed the wild-type DNA polymerase and the Y265C mutator mutant in mouse LN12 cells, which have endogenous DNA polymerase beta activity. We demonstrate that expression of the Y265C mutator mutant in the LN12 cells results in an 8-fold increase in the spontaneous mutation frequency of lambdacII mutants compared with expression of the wild-type protein. Expression of Y265C results in at least a 40-fold increase in the frequency of deletions of three bases or more and a 7-fold increase in point mutations. Our results suggest that the mutations we observe in vivo result directly from the action of the mutator polymerase. To our knowledge, this is the first demonstration of a mutator phenotype resulting from expression of a DNA polymerase mutator mutant in mammalian cells. This work raises the possibility that variant polymerases may act in a dominant fashion in human cells, leading to genetic instability and carcinogenesis.