Certain particulate compounds of hexavalent chromium are well-known occupational and environmental human carcinogens. Hexavalent chromium primarily enters the cells and undergoes metabolic reduction; however, the ultimate trivalent oxidation state of chromium, Cr(III), predominates within the cell. DNA-bound Cr(III) has been previously shown to decrease the fidelity of replication in the M13 phage mutation assay. This study was done to understand how Cr(III), in the presence of physiological concentrations of magnesium, affects the kinetic parameters of steady-state DNA synthesis in vitro across site-specific O6-methylguanine (m6dG) residues by DNA polymerase beta (pol beta). Cr(III) binds to the short oligomer templates in a dose-dependent manner and stimulates the activity of pol beta. Cr(III) stimulates the mutagenic incorporation of dTTP opposite m6dG more than the nonmutagenic incorporation of dCTP, and thereby Cr(III) further decreases the fidelity of DNA synthesis across m6dG by pol beta. In contrast, Cr(III) does not affect the fidelity of DNA synthesis across the normal template base, dG. Both the enhanced activity and the mutagenic lesion bypass in the presence of Cr(III) may be associated with Cr(III)-dependent stimulation of pol beta binding to DNA as reported here. This study shows some of the mechanisms by which mutagenic chromium affects DNA synthesis.