Leu-enkephalin, at concentrations between 18 microM and 1.8 pM, was administered in a single or daily dose to dissociated mesencephalic raphe cell cultures maintained for 3 or 5 days. Daily administration of Leu-enkephalin produced an inhibition of high affinity uptake of [3H]5-HT, a measure of serotonergic process outgrowth in cultures of fetal neurons. This inhibition was maximal at a dose of 18 nM in both 3 (59%, P less than 0.05)- and 5 (38%, P less than 0.05)-day cultures. The expression of uptake was consistently lower in 5-day cultures than in 3-day cultures at all concentrations tested. In marked contrast, a single dose of Leu-enkephalin at the time of plating stimulated uptake in 3- and 5-day cultures. Maximal stimulation was observed at 180 nM for both 3 (191%, P less than 0.05)- and 5 (140%, P less than 0.05)-day cultures. The results obtained after a single dose of the opioid may reflect a paradoxical stimulation probably due to a rebound mechanism of receptors since co-administration of bacitracin (0.5 mg/ml), an aminopeptidase inhibitor, resulted in inhibition of the uptake expression. Together these results indicate that Leu-enkephalin can function as an inhibitory regulatory growth factor for neuronal cultures when constant exposure to this opioid is maintained over time.