Rat liver and brain alpha 1-adrenergic receptors were purified 500 fold by successive chromatographic steps using heparin- and wheat germ agglutinin-agarose; an affinity matrix constructed by coupling CP85.224 (a derivative of prazosin) to affigel 102. It is shown that the existence in brain of an alpha 1-adrenergic receptor subpopulation, which is structurally distinct from that previously characterized. Chlorethylclonidine, irreversibly inactivates [3H] prazosin binding sites in partially purified membrane preparations of rat liver. Under identical conditions, only 50% of receptors are irreversibly inactivated. Computer modelling of data obtained from the competition by the alpha-antagonists, WB 4101 and phentolamine, for [3H] prazosin binding to partially purified preparations of rat liver is best fit by assuming a single low-affinity site for both ligands. However, the partially purified brain preparations indicates the presence of two affinity binding sites for these antagonists. Prior alkylation of brain receptors with chlorethylclonydyne results in the loss of the low-affinity phentolamine and WB4101 binding sites. These data provide evidence for the existence of a single receptor subpopulation (alpha 1b) in rat liver and for two subpopulations (alpha 1a and alpha 1b) in rat brain. The significance of these results in understanding the signal mechanisms which allow cellular responsiveness to alpha 1-adrenergic receptor agonists is discussed.