In vitro binding experiments were performed to determine whether known subtypes of the putative sigma receptor exhibit a differential distribution across brain regions and species. Rat brains were dissected into nine regions, pooled, and used to prepare membranes for ligand binding studies. Whole guinea pig brains were prepared in an identical manner for comparison to rat. sigma 1 Receptors were labeled with [3H](+)-pentazocine. sigma 2 Receptors were labeled with [3H]1,3-di-o-tolylguanidine (DTG) in the presence of 1 microM dextrallorphan to mask sigma 1 sites. Non-specific binding was determined in the presence of 10 microM haloperidol. Filtration and scintillation spectroscopy provided the binding values. The experiment revealed marked variation in the ratio of sigma 2 to sigma 1 binding across brain regions ranging from a low of 1.63 in the hindbrain to 3.51 in the cerebellum, that result mainly differences in the density of the receptors. Scatchard analysis on membranes derived from the hindbrain and cortex suggested that the effects were due primarily to regional differences in densities of receptor subtypes rather than different affinities. Guinea pig brain showed a marked preponderance of sigma 1 receptors with a ratio (sigma 2/sigma 1) of 0.67. These findings demonstrate that sigma 1 and sigma 2 receptors are differentially distributed in rat brain.