These studies define the site and mechanisms of reproductive toxicity of ethylene glycol monomethyl ether (EGME) in a nongravid female animal model using in vivo and in vitro methods. In vivo studies assessed vaginal cytology and histology, ovarian histology, and serum hormones in 80- to 90-day-old, adult, regularly cycling, female Sprague-Dawley rats treated daily with EGME or vehicle by oral gavage. Dose-response and time-course studies (four to nine rats per group per treatment) determined that 300 mg/kg EGME suppressed cyclicity without systemic toxicity within 3 to 8 days, and doses less than 100 mg/kg had no effect. Pathogenesis studies (six to nine rats per time and treatment) determined that 300 mg/kg EGME elevated serum progesterone within 32 hr after dosing, while serum estradiol, FSH, LH, and prolactin remained at baseline levels. In EGME-treated rats, cyclicity was suppressed, ovulation was inhibited, and corpora lutea were hypertrophied. Thus, EGME appeared to target the ovarian luteal cell. To further examine the toxicity in vitro, luteal cells were recovered from 23-day-old, hCG-primed Sprague-Dawley rats and treated with 0-10 mM methoxy acetic acid (MAA), the proximate toxic metabolite of EGME. MAA (1-10 mM) maintained elevated progesterone levels as production declined in untreated cells at 24 and 48 hr of culture. Progesterone production was maintained independent of LH-stimulated cAMP levels. MAA decreased ATP, but only at 48 hr and at 2.5 mM or greater concentrations. Thus, these studies establish that the ovarian luteal cell is a target of EGME and MAA in vivo and in vitro and that the effect on luteal cell progesterone production is likely independent of LH-stimulated cAMP pathways.