The developmental expression of neuropeptide Y (NPY) and leucine- enkephalin (L-Enk) was examined in embryonic, early postnatal, and adult chromaffin cells with double- and triple-label immunocytochemical techniques and compared to the expression of immunoreactivity for tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT). In addition, the establishment of preganglionic innervation was assessed by labeling for choline acetyltransferase (ChAT) and L-Enk. NPY-IR was detectable on embryonic (E) day 15 in a clustered subpopulation of TH-IR cells. L-Enk and PNMT-IR cells were initially present on E16 in a separate nonclustered population of TH-IR cells. By late embryonic development, twice as many TH-IR cells expressed NPY and 4 times as many expressed L-Enk as in the adult. In contrast to early embryonic development, NPY-IR was evident in both the clustered and nonclustered subpopulation of TH-IR cells at this time. The proportion of NPY-IR chromaffin cells decreased to adult values during the first postnatal week at the time when obviously clustered TH-IR cells were no longer observed. The embryonic rise in the proportion of L-Enk-IR cells correlates with the developmental increase in glucocorticoid production, while the postnatal decrease corresponds to the appearance of ChAT-IR in the preganglionic innervation of the adrenal medulla. These results indicate that NPY and L-Enk are expressed at different times and in different subpopulations of cells in the embryonic adrenal. Further, the observation that peptide expression by chromaffin cells undergoes marked changes during development raises the possibility that a number of factors including developmental history, environmental signals and impulse activity play a role in the regulation of neuropeptide expression in sympathoadrenal derivatives of the neural crest.