The characteristics of the metabolism of bis(2-methoxyethyl)ether (111966) (diglyme) to 2-methoxyethanol (109864) (2ME) were explored in this study. Male Sprague-Dawley-rats were pretreated with phenobarbital (PB), beta-naphthoflavone (beta-NF), ethanol, or diglyme and were then sacrificed. Hepatic microsomes were isolated and incubated. In microsomal incubations containing an NADPH generating system, three major metabolites were detected; 2ME, 2-(2- methoxyethoxy)ethanol (111773), and an unknown compound. In analysis of microsomes of untreated rats, less than 2% of the added diglyme was metabolized after 30 minutes. The activity of aniline- hydroxylase, a cytochrome-P-450 inducer, increased following ethanol pretreatment. The activities of various cytochrome-P-450 enzymes, as well as the metabolism of diglyme, increased with PB or ethanol pretreatment. Pretreatment with diglyme increased microsomal levels of cytochrome-P-450 by 70% over control microsomes. 2ME formation was dependent on both the incubation time and the concentration of microsomal proteins present. Various cytochrome-P-450 inhibitors reduced the metabolism of diglyme. The inhibitor isoniazid specifically inhibited the isozyme P450IIE1, even in diglyme or ethanol pretreated microsomes. Human hepatic microsomes also metabolized the NADPH dependent breakdown of diglyme to 2ME. A significantly positive correlation was found between aniline- hydroxylase activity and 2ME formation in both rats and humans. Due to the extensive increases in metabolism after pretreatment with diglyme, diglyme was implicated as an inducer of cytochrome-P-450. The authors conclude that the central ether linkage of diglyme is cleaved by ethanol, PB and isozymes of cytochrome-P-450, most notably P450IIE1.