Changes of cyclic AMP levels were used to assess the specificity of the beta-adrenergic receptor in primary cultures of cells prepared from the intermediate lobe of rat pituitary gland. During a 4 min incubation, beta-adrenergic agonists led to a 4 to 6 fold stimulation of cyclic AMP concentration with the following order of potency (Kd values): zinterol (0.75 nM) greater than hydroxybenzylisoproterenol (1.0 nM) greater than (--)-isoproterenol (4.6 nM) greater than soterenol greater than (7.7 nM) greater than (--)-epinephrine (10 nM) greater than OPC 2009 (procaterol, 11 nM) much greater than (--)-norepinephrine (300 nM). The potent antagonists cyanopindolol, (--)-propranolol and hydroxybenzylpindolol reversed the stimulatory effect of (--)-isoproterenol at Kd values of 0.4-0.6 nM. Other beta-adrenergic antagonists had the following order of potency: pindolol = (--)-alprenolol = timolol (0.9-1.0 mM) much greater than metoprolol (100 nM) greater than dichloroisoproterenol (300 nM) greater than butoxamine (1100 nM). The beta 1-selective antagonist practolol had a low potency at 700 nM. The stereoselectivity of the receptor is indicated by the 400 to 70 fold higher potency of the (--)-isomers of isoproterenol, epinephrine and propranolol as compared to their (+)-stereoisomers. The data show that the beta-adrenergic receptor in the intermediate lobe of the rat pituitary gland is mainly of the bet 2-subtype. Study of this pure population of postsynaptic beta-adrenergic receptors where binding could be correlated with other parameters of cellular activity (cyclic AMP formation and alpha-MSH secretion) should yield useful information about the less accessible adrenergic systems of the brain.