Rat hepatic microsomes catalysed the NADPH-dependent cleavage of the central ether linkage of bis(2-methoxyethyl) ether (diglyme) yielding 2-methoxyethanol (2ME). Microsomes isolated from phenobarbital- or ethanol-pretreated rats exhibited an increased capacity to cleave diglyme to 2ME. This ethanol-induced increase in 2ME formation was not observed if incubations contained the cytochrome P450IIE1 inhibitor isoniazid. Pretreatment of rats with diglyme significantly increased microsomal P-450 levels, P-450-associated enzyme activities and the conversion of diglyme to 2ME. Following the diglyme pretreatment, an almost 30-fold increase in pentoxyresorufin dealkylase activity (P450IIB1/2) was evident in rat hepatic microsomes. Human hepatic microsomes also catalysed the NADPH-dependent cleavage of diglyme to 2ME. The formation of 2ME from diglyme correlated with the aniline hydroxylase activity (P450IIE1) levels measured in human hepatic microsomes. Studies using microsomes isolated from a cell line transfected with specific human P-450 cDNAs indicate that human CYP2E1 catalyses the conversion of diglyme to 2ME. These results suggest that the central ether linkage of diglyme is cleaved by rat and human P-450 and the specific involvement of hepatic P450IIE1 in this process is implicated.