The nematocide 1,2-dibromo-3-chloropropane (DBCP) was converted to products which are mutagenic for Salmonella typhimurium TA 100 in the presence of rat liver microsomes, NADPH, and oxygen. Typical in vivo and in vitro inhibitors of cytochrome P-450 decreased DBCP mutagenicity in the presence of microsomes. Addition of glutathione to cytosolic preparations failed to bioactivate DBCP to mutagenic metabolites. Mutagenicity studies with selectively deuterated analogs showed that substitution of deuterium for hydrogen at C-1 or C-3 of DBCP modestly decreased mutagenicity, but that deuteration at both C-1 and C-3 markedly decreased mutagenicity. The formation rates of the potent direct-acting mutagen, 2-bromoacrolein (2-BA), in incubations of DBCP and its deuterated analogs with rat liver microsomes, correlated with the isotope effects on mutagenicity. Characterization of 2-BA was accomplished by gas chromatography-mass spectrometry using positive-ion chemical ionization. Mass spectral analysis of 2-BA formed from specifically deuterated analogs of DBCP indicated that initial oxidative dehalogenation at C-1 followed by a spontaneous beta-elimination reaction was the preferred pathway in the formation of 2-BA from DBCP. These results demonstrate that mutagenic metabolites of DBCP are formed by cytochrome P-450-mediated oxidative metabolism, and that 2-BA is a major mutagen formed.