[3H](+/-)Carazolol, a newly available beta-adrenergic receptor antagonist, can be used to characterize beta-adrenergic receptor subtypes present in membrane vesicles derived from canine ventricular myocardium and canine lung. [3H](+/-)Carazolol binding is saturable, of high affinity, and is displaceable by beta-adrenergic agents in accordance with their known pharmacological potencies. The interaction of carazolol with beta-adrenergic receptors is stereospecific; the (-) stereoisomer demonstrates greater potency than the (+) stereoisomer. Kinetic analysis of [3H](+/-)carazolol interaction with beta-adrenergic receptors suggests that presence of two phases of interaction, consistent with initial rapidly reversible "low" affinity association of ligand with receptor, followed by isomerization to form a high affinity, slowly reversible complex. Through use of a [3H](+/-)carazolol binding assay based on the high affinity complex, pharmacological specificities of beta-adrenergic receptor populations of canine myocardium and lung were quantified. Analysis using computer-assisted techniques suggests a beta 1/beta 2 receptor ratio of approximately 85%/15% for canine myocardium and 5%/95% for canine lung. In the absence of added guanine nucleotides, comparison of potencies of beta-adrenergic agonists in the two membrane systems suggests significant beta 2 selectivity of l-isoproterenol and l-epinephrine, and non-selectivity of norepinephrine. In the presence of saturating levels of guanine nucleotides, comparison of agonist potencies confirms the non-selectivity of l-isoproterenol and l-epinephrine, and beta 1 selectivity of norepinephrine. These results demonstrate that the state of guanine nucleotide regulation of receptors should be defined when examining agonist selectivities for beta-adrenergic receptor subtypes in vitro.