The effect of prenatal exposure to methadone via maternal osmotic minipumps was studied on brain regional acetylcholine (ACh) turnover and dopamine (DA), norepinephrine (NE), serotonin (5-hydroxytryptamine, 5-HT) and their metabolites in 21-day-old female and male rats. ACh content was not affected in any region studied. However, the turnover rate of ACh (TRAch) was increased significantly in the striata and parietal cortices of both sexes. Two gender-specific changes were observed: a profound decrease in hypothalamic TRACh in the females and an increase in hippocampal TRACh in the males. No changes were observed in TRACh in the medulla-pons or the frontal cortex of either sex. The reduction in TRACh was accompanied by a threefold increase in DA content in the hypothalamus of the methadone-exposed females. No other changes were observed in DA, NE, or 5-HT, save for increased 5-HT content in the medulla-pons of the male methadone-exposed rats. Thus, prenatal methadone exposure produces several lingering changes in cholinergic function, many of which were not apparent in the immediate postnatal period. Although striatal ACh content was no longer reduced in methadone-exposed rats, striatal cholinergic function remains disrupted. It remains to be proven whether these differences are a direct effect of methadone exposure or are a consequence of neonatal withdrawal.