The effect of prenatal exposure to methadone via maternal osmotic minipumps on the expression of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) has been studied by light microscopy in the striatum of male and female rats. At postnatal day 10, rats of both sexes exhibit reduced intensity of ChAT-immunoreactive staining in striatal neurons in the methadone-treated group in comparison to either untreated or water-treated controls. Although the number and distribution of ChAT-immunoreactive neurons appear to be similar across all three groups, the size (cross-sectional area) of these neurons is significantly smaller in the methadone-treated animals. By postnatal day 22, there are no differences in the ChAT immunoreactivity of striatal neurons between the water-treated and methadone-treated animals. The size of these neurons in female animals of both treatment groups is significantly larger than those of male animals in the same groups at postnatal day 22. AChE histochemical staining is qualitatively similar between treatment groups in both age groups. Thus, prenatal exposure to methadone appears to produce a delay in the expression of ChAT in striatal neurons. It remains to be demonstrated whether these differences are a direct effect of methadone exposure or are a consequence of neonatal withdrawal.