A number of investigators have shown compelling evidence for multiplicity of benzodiazepine (BDZ) receptors. The present study addresses the query of BDZ receptor heterogeneity, in vitro, with respect to temperature. In competition studies involving rat cerebellar tissue, CL 218,872 produced Hill slopes near unity at both 0°C and 37°C. In contrast, similar experiments utilizing cortical tissue from rats and mice produced Hill slopes of 0.69 and 0.66 at 0°C and 37°C respectively. 3H-Flunitrazepam-photoaffinity labeling of cortical and cerebellar membranes was conducted at 0°C and 37°C. SDS-PAGE fluorographic analyses of photolysed 3H-flunitrazepam ( 3H-Flu) revealed one intensely labeled 51K band in the cerebellum at both temperatures, which was specifically chased by diazepam. Similar experiments conducted in cortical tissue revealed photoaffinity labeling of at least three distinct macromolecules, one intense 51K and two less intense 55K and 59K bands. Labeling of each of these bands was chased specifically by diazepam. These data, taken together, indicate the existence of regional BDZ receptor heterogeneity under physiological conditions.