alpha 1-Adrenergic receptor subtypes were differentiated by their affinities for the competitive antagonist WB 4101 and their sensitivities to inactivation by chlorethylclonidine (CEC) in eight rat brain regions. WB 4101 showed low Hill coefficients for inhibition of specific 125I-[2-beta-(4-hydroxyphenyl)ethylaminomethyl]tetralone (125IBE) binding in all regions. Nonlinear regression analysis showed that there were two binding sites with different affinities for WB 4101 in each region. The proportions of these sites varied among regions, although the affinity of WB 4101 for each site remained constant. Thalamus and cerebral cortex had the highest proportion of low-affinity sites, whereas hippocampus and pons-medulla had the highest proportion of high-affinity sites. Pretreatment with CEC in hypotonic buffer significantly reduced the density of 125IBE binding sites in all brain regions. Cerebral cortex and cerebellum had the highest proportion of CEC-sensitive sites, whereas hippocampus and spinal cord had the highest proportion of CEC-insensitive sites. There was a significant correlation between the proportion of binding sites with a low affinity for WB 4101 and those sensitive to inactivation by CEC.