Affinity-purified antibodies raised against the peptide sequence H3 (349-358) receptor specifically recognized two protein species with Mr 62,000 and 93,000 in adult mouse forebrain membranes. Both immunoreactive species were suppressed greatly by preincubation of the antibody with the respective peptide. Immunohistochemical analysis using affinity-purified anti-H3 (349-358) antibodies yielded a high degree of coincidence with ligand-autoradiographical information, with high levels detected in the CA3 and dentate gyrus of the hippocampus, laminae V of the cerebral cortex, the olfactory tubercle, Purkinje cell layer of the cerebellum, substantia nigra, globus pallidus, thalamus and striatum. This study suggests further biochemical evidence for multiple H3 receptor subtypes and the widespread distribution of the H3 receptor in the mammalian brain.