The coupling of the pharmacologically defined alpha 1A- and alpha 1B-adrenoceptors to the hydrolysis of phospho[3H]inositides (PI) was investigated in ventricular myocytes freshly isolated from adult rat hearts. The alpha 1-adrenoceptor population in the heart was characterized by competitive binding experiments using [3H]prazosin and the alpha 1A-adrenoceptor-selective antagonist 5-methyl urapidil. It was heterogeneous with approximately 25% being pharmacologically of the alpha 1A-adrenoceptor subtype and 75% being of the alpha 1B-adrenoceptor subtype. Epinephrine, norepinephrine, or phenylephrine stimulated PI hydrolysis in the presence or absence of propranolol. The greatest stimulation (7-fold) was with epinephrine. The half-maximum effective concentrations for agonists were approximately 0.5-3.5 and 0.2 microM in the absence and presence of propranolol, respectively. The inhibition by 5-methyl urapidil of the stimulation of PI hydrolysis by a fixed concentration of epinephrine fitted a two-site competition curve. The distribution between high-affinity (25%) and low-affinity (75%) sites suggested that both the alpha 1A- and alpha 1B-adrenoceptors were coupled to PI hydrolysis in proportion to their relative abundance. Equally, the stimulation of PI hydrolysis by epinephrine in the presence of a fixed concentration of 5-methyl urapidil was biphasic. In addition, chloroethylclonidine, an irreversible inhibitor of the alpha 1B-adrenoceptor, inhibited the epinephrine stimulation of PI hydrolysis by 35%. We conclude that the pharmacologically defined alpha 1A- and alpha 1B-adrenoceptor subtypes are both coupled to PI hydrolysis in the ventricular myocyte.