kappa opioid receptors (kappa receptors) have been characterized in homogenates of guinea pig and rat brain under in vitro binding conditions. kappa receptors were labeled by using the tritiated prototypic kappa opioid ethylketocyclazocine under conditions in which mu and delta opioid binding was suppressed. In the case of guinea pig brain membranes, a single population of high-affinity kappa opioid receptor sites (kappa sites; Kd = 0.66 nM, Bmax = 80 fmol/mg of protein) was observed. In contrast, in the case of rat brain, two populations of kappa sites were observed--high-affinity sites at low density (Kd = 1.0 nM, Bmax = 16 fmol/mg of protein) and low-affinity sites at high density (Kd = 13 nM, Bmax = 111 fmol/mg of protein). To test the hypothesis that the high- and low-affinity kappa sites represent two distinct kappa receptor subtypes, a series of opioids were tested for their abilities to compete for binding to the two sites. U-69,593 and Cambridge 20 selectively displaced the high-affinity kappa site in both guinea pig and rat tissue, but were inactive at the rat-brain low-affinity site. Other kappa opioid drugs, including U-50,488, ethylketocyclazocine, bremazocine, cyclazocine, and dynormphin (1-17), competed for binding to both sites, but with different rank orders of potency. Quantitative light microscopy in vitro autoradiography was used to visualize the neuroanatomical pattern of kappa receptors in rat and guinea pig brain. The distribution patterns of the two kappa receptor subtypes of rat brain were clearly different. The pattern of rat high-affinity kappa sites paralleled that of guinea pig in the caudate-putamen, mid-brain, central gray substance of cerebrum, and substantia nigra; interspecies differences were apparent throughout most of the rest of the brain. Collectively, these data provide direct evidence for the presence of two kappa receptor subtypes; the U-69,593-sensitive, high-affinity kappa 1 site predominates in guinea pig brain, and the U-69,593-insensitive, low-affinity kappa 2 site predominates in rat brain.