Developmental changes in rat adrenal [Met5]enkephalin (ME) immunoreactivity and [Met5]enkephalinargininephenylalanine (MERF) immunoreactivity (which presumably reflects proenkephalin) were examined. The concentration of MERF appeared highest at the end of the first week postnatally and decreased fourfold to near adult values by day 21, while ME levels decreased only about twofold. This indicates a fall in the adrenal proenkephalin:ME ratio during the transition from the suckling to the weanling period in the rat. The elevated MERF levels during the suckling period may result from deficient processing of proenkephalin to free ME. An increase in the activity of the processing enzyme carboxypeptidase E was observed at day 15, which may contribute to the developmental fall in the adrenal proenkephalin to free ME ratio. Mimicking sympathetic activity by nicotine treatment (3 mg/kg i.p. twice daily from birth) produced a precocious decrease in the MERF:ME ratio at day 8, and also increased the activity of the processing enzyme. Denervation at day 10 markedly decreased adrenal ME and produced a three-fold increase in MERF:ME ratio when measured at day 30. Adrenal carboxypeptidase E activity was also decreased after denervation. In summary, these results suggest that increasing preganglionic sympathetic nerve activity during the third week postnatally stimulates proenkephalin processing and leads to the developmental decrease in MERF:ME ratio in the rat adrenal.