Antisera against neuropeptide Y, [Met]enkephalin and bombesin were used for characterizing the immunoreactive material in subcellular fractions of bovine adrenal medulla. Neuropeptide Y was identified by high performance liquid chromatography and by immunoblotting. Subcellular fractionation established that neuropeptide Y is present in chromaffin granules. During stimulation of the adrenal it is released concomitantly with catecholamines. The soluble proteins of chromaffin granules contain 1.9 micrograms neuropeptide Y/mg protein which gives 429 copies of neuropeptide Y for a single granule. In two-dimensional immunoblots two peptides of the same molecular size, but with differing pI (6.4 and 7.3) react with the antiserum against neuropeptide Y. There was no evidence for the presence of a larger neuropeptide Y precursor in chromaffin granules. On the other hand, larger enkephalin-containing peptides could be detected by immunoblotting. The subcellular distribution of these enkephalin precursors differed. The larger peptides (23.3 and 18.2 kD) were more concentrated in lighter granules when compared to the smaller precursors (12.6 and 8.6 kD) which is consistent with proteolytic processing of these peptides during granule maturation. An antiserum against bombesin reacts in immunoblots with the chromogranin B family. This study further illustrates that chromaffin granules contain a complex mixture of neuropeptide-immunoreactive material. The combination of immunoblotting with subcellular fractionation appears as a useful tool to characterize these peptides and their precursors.