The specific melanotropin (MSH) binding sites of rat lacrimal glands were characterized with respect to anatomic distribution, peptide specificity and selectivity, and coupling to a biological response. Tissue distribution of MSH binding sites was determined by autoradiography following in situ binding of a radiolabeled, biologically active preparation of a superpotent alpha-MSH analog, [125I]-[Nle4,D-Phe7]-alpha-MSH ([125I]-NDP-MSH). Intense, specific (i.e., alpha-MSH-displaceable) [125I]-NDP-MSH binding was observed throughout lacrimal acinar tissue, but not in ducts or stroma. In freshly isolated lacrimal acinar cells, specific binding of [125I]-NDP-MSH was maximal within 30 min and rapidly reversible, with a dissociation half-time of about 15 min. A number of melanotropins [alpha-MSH, [N,O-diacetyl-Ser1]-alpha-MSH, [des-acetyl-Ser1]-alpha-MSH, beta-MSH, ACTH(1-24) and ACTH(1-39)] were recognized by these binding sites, as assessed by their inhibition of [125I]-NDP-MSH binding; NDP-MSH was the most potent (IC50 = 1.3 x 10(-9) M). In contrast, other peptides, including ACTH(4-10) and the nonmelanotropic peptides VIP, substance P, somatostatin, and ACTH(18-39) (CLIP), had no effects on tracer binding. In isolated lacrimal acinar cells, alpha-MSH and NDP-MSH stimulated intracellular cyclic AMP accumulation. We conclude that lacrimal acinar cells express functional receptors recognizing melanotropins, suggesting that the lacrimal gland may be a target for physiological regulation by endogenous melanotropins.