The metabolism of the reproductive and developmental toxicant bis(2-methoxyethyl)ether (diglyme) was studied in isolated rat hepatocytes and in the intact rat. Male Sprague-Dawley rats (190–220 g) were used in both studies. Hepatocytes, isolated by a two-step in situ collagenase perfusion of the liver, were cultured as monolayers and incubated with [¹⁴C]diglyme at 1, 10, 30, and 50 μM for up to 48 h. For the in vivo study, rats were given single oral doses of [¹⁴C]diglyme at 5.1 mmol/kg body wt, and urine was collected for up to 96 h. Radioactive compounds in the culture medium or in the urine were separated by high performance liquid chromatography and quantified with an in-line radioactivity monitor. Metabolites were identified by comparison of their chromatographic retention times and their mass spectra with those of authentic compounds. The principal metabolite from hepatocytes and in the urine was (2-methoxyethoxy)acetic acid (MEAA). This metabolite accounted for approximately 36% of the radioactivity in the 48-h culture medium and about 67% of the administered dose in the 48-h urine. Other prominent metabolites common to both systems included 2-(2-methoxyethoxy)ethanol, methoxyacetic acid (MAA), 2-methoxyethanol, and diglycolic acid. The diglyme metabolite profiles from urine and from hepatocytes were qualitatively similar, demonstrating that, in the rat, hepatocytes serve as a good model system for predicting the urinary metabolites of diglyme. Moreover, MEAA was shown to be the metabolite best suited for use as a short-term biological marker of exposure to diglyme. Pretreatment of rats with ethanol resulted in a marked increase in the overall in vitro metabolism of diglyme. The major metabolic pathways for diglyme involve O-demethylation and cleavage of the central ether bond, and it is the latter pathway that leads to the formation of MAA, the metabolite associated with the reproductive and developmental toxicity of diglyme. The amounts of MAA formed in hepatocytes from ethanol-pretreated rats ranged from two to four times those formed in hepatocytes from untreated rats.