In PC12 rat pheochromocytoma cells differentiated with nerve growth factor (NGF), neuropeptide Y inhibited depolarization-stimulated catecholamine synthesis as determined by in situ measurement of 3,4-dihydroxyphenylalanine (DOPA) production in the presence of the decarboxylase inhibitor m-hydroxybenzylhydrazine (NSD-1015). The inhibition by neuropeptide Y was concentration-dependent and was prevented by pretreatment with pertussis toxin, suggesting the involvement of a GTP-binding protein of the Gi or Go subtype. The neuropeptide Y analog [Leu31,Pro34]neuropeptide Y also caused inhibition of DOPA production, but was less potent than neuropeptide Y itself, while peptide YY and neuropeptide Y-(13-36) had no significant effect. This pattern is most consistent with the involvement of the neuropeptide Y Y3 receptor subtype. In PC12 cells differentiated with dexamethasone, neuropeptide Y also caused a concentration-dependent inhibition of DOPA production, while peptide YY was again without effect. Neuropeptide Y had no effect on DOPA production in undifferentiated PC12 cells. These results indicate that neuropeptide Y can modulate catecholamine synthesis in addition to its modulatory effects on catecholamine release.