The biosynthesis of the hormone melatonin (MEL) by the mammalian pineal gland has been thought to be regulated strictly by stimulatory factors, most predominantly norepinephrine (NE), released from the sympathetic nerve fibers which heavily innervate the gland. Evidence from many investigators suggests that sympathetic fibers may colocalize other neuroactive factors in addition to NE. One of these factors is neuropeptide Y (NPY), which has been found in the nerve fibers of the pineal gland. The present study sought to explore potential interactions between NE and NPY in the regulation of pineal MEL secretion. Specific, saturable, and reversible binding of 125I-NPY to intact cultured pinealocytes was measured with an affinity constant of 1 nM and an NPY binding site density of 0.04 pmol/mg of protein. In addition, cell culture studies revealed that NPY represents a potent (IC50 of 0.4 nM) endogenous inhibitor of NE-stimulated MEL secretion. However, this inhibition is accompanied by only a modest reduction (35%) of cyclic AMP accumulation. These findings reinforce the view that the mammalian pineal gland, which appears to integrate both inhibitory as well as stimulatory signals, is an important model of autonomic function, particularly in the context of biological rhythmicity.