The existence of multiple brain melanocortin receptor types has been postulated, based on the complex pharmacology of intracerebrally administered melanocortin (melanocyte-stimulating hormone-related) peptides. In this study, this hypothesis was tested by determining whether different brain melanocortin receptor populations can be discriminated on a pharmacologic or neuroanatomic basis. The abilities of various pharmacologically active native melanocortins and structural analogs, as well as other test substances, to compete with biologically active [125I]Nle4,D-Phe7-alpha-MSH ([125I]NDP-MSH) for binding to melanocortin receptors was determined, by in vitro binding and autoradiography in frozen rat brain tissue sections. We have previously shown that native melanocortins including alpha-MSH, gamma-MSH and ACTH1-39 compete with [125I]NDP-MSH for binding to brain tissue sites. In the present studies, each of the melanocortin peptides alpha-MSH, des-acetyl-alpha-MSH, beta-MSH and ACTH1-24 when present at 1 microM virtually eliminated [125I]NDP-MSH binding in each of a series of brain structures, including medial preoptic area, caudate putamen, olfactory tubercle, bed nucleus of the stria terminalis, ventral part of the lateral septal nucleus, hypothalamic periventricular and paraventricular nuclei, dorsal anterior amygdaloid area, substantia innominata and thalamic paraventricular nucleus; as well as in extraorbital lacrimal gland, a peripheral melanocortin target. In contrast, the behaviorally and neurotrophically active melanocortin analogs Met(O2),D-Lys,Phe9-alpha-MSH4-9 (Org2766), ACTH4-9, and the antipyretic peptide alpha-MSH11-13 did not affect [125I]NDP-MSH binding at concentrations up to 100 microM, implying that the receptors or receptor binding sites which mediate the actions of these analogs must comprise additional types, distinct from those which bind [125I]NDP-MSH. Binding of [125I]NDP-MSH was also unaffected by the nonmelanotropic peptides ACTH1-4, ACTH34-39 and vasoactive intestinal polypeptide (VIP) and by the antipyretic drugs acetaminophen and lysine-salicylate. Although some of the brain structures are known to express mRNA encoding a gamma-MSH-preferring melanocortin receptor type known as MC3, the relative order of binding affinities of melanocortins, determined in concentration-response studies, was NDP-MSH > or = ACTH1-24 > or = alpha-MSH > gamma-MSH > ACTH4-10 in all brain structures. This suggests that other melanocortin receptor type(s) in addition to MC3 probably account for most of the [125I]NDP-MSH binding detectable in the brain.(ABSTRACT TRUNCATED AT 400 WORDS)