We studied the effects of the 36-amino acid peptide, neuropeptide Y (NPY), on salt secretion by the rectal gland of Squalus acanthias. We used three preparations: whole isolated perfused glands, freshly prepared separated rectal gland tubules, and confluent monolayers of cultured rectal gland cells. In perfused glands NPY inhibited secretion stimulated by vasoactive intestinal peptide (VIP), forskolin, or adenosine 3',5'-cyclic monophosphate (cAMP) and theophylline. Maximal inhibition of 63 +/- 3.4% was seen at 3 x 10(-8) M NPY, with half-maximal effect at 3 x 10(-9) M. NPY did not inhibit the basal activity of rectal gland adenylate cyclase or that stimulated by VIP. The inhibitory action of NPY was not prevented by procaine, nifedipine, or diltiazem, suggesting that it was not secondary to the release of somatostatin or other unknown neurotransmitters from rectal gland nerves. In confirmation, somatostatin was not detected in the venous effluent after administration of NPY. NPY also inhibited transport-related oxygen consumption in separated rectal gland tubules and inhibited short-circuit current generated by confluent monolayers of primary cultures of rectal gland cells. The results indicate that NPY inhibits chloride secretion by a direct action on cells of the shark rectal gland at a site distal to the generation of cAMP.