The presence of alpha 1- and alpha 2-receptor subtypes were studied in steer stalk-median eminence (SME) using a selective alpha 1-ligand, [3H]-prazosin, and a selective alpha 2-ligand, [3H]-clonidine. Scatchard analysis of the equilibrium isotherms of specific [3H]-prazosin binding indicated a single class of high affinity (Kd = 0.36 +/- 0.05 nM) and low capacity (Bmax = 111 +/- 19 fmol/mg protein) binding sites, which accounted for approximately 26% of the total sites identified by the non-subtype-selective-alpha-antagonist, dihydroergocryptine ([3H]-DHE). Similarly, computer modeling of the biphasic competition curve of prazosin displacing [3H]-DHE also revealed that there are approximately 26% high affinity sites, presumably alpha 1-, and approximately 74% low affinity sites, presumably alpha 2-receptors in the steer SME. A scatchard plot of specific [3H]-clonidine binding was biphasic and was resolved into a high (Kd = 1.60 +/- 0.34 nM) and low (Kd = 34.5 +/- 7.85 nM) affinity site with the binding capacities of 43 +/- 5 and 200 +/- 37 fmol/mg protein, respectively. The total number of alpha 2-sites identified by [3H]-clonidine accounted for approximately 58% of the total sites labeled by [3H]-DHE, which was in good agreement with that calculated from the competition of clonidine for [3H]-DHE binding (approximately 65% alpha 2 and approximately 35% alpha 1). The rank orders of potency of alpha-adrenergic agents to compete for sites labeled by [3H]-prazosin and [3H]-clonidine were consistent with their labeling alpha 1- and alpha 2-subtypes, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)