Methadone was administered in the form of a slow release emulsion to nonpregnant rats and pregnant animals at early, middle, or late gestation. Following a 48-h treatment period, plasma, urine, and amniotic fluid were analyzed for methadone and its two major metabolites using solid-phase extraction and reversed-phase HPLC. Methadone and its primary metabolite were detected in plasma and urine of all rats with levels of both compounds comparable at all stages of gestation. No significant difference was found for either methadone or its primary metabolite between gestation groups and nonpregnant animals. Amniotic fluid collected in middle and late gestation groups contained methadone and primary metabolite at levels similar to that of maternal plasma. These data show that methadone can be detected in plasma, urine, and amniotic fluid following short-term treatment of pregnant rats with the opiate. Methadone levels did not alter during pregnancy and were, in fact, similar to that of nonpregnant animals. These results indicate that the fetal environment contains methadone levels comparable to that of the maternal circulation.