There are specific phorbol ester receptors on cardiac myocytes which may be identical with the calcium/phospholipid-dependent protein kinase (protein kinase C). Incubation of enzymatically dissociated rat cardiac myocytes with biologically active phorbol esters (such as 4 beta-phorbol-12, 13-dibutyrate and 12-O-tetradecanoyl phorbol-13-acetate) leads to a time- and concentration-dependent loss of beta-adrenergic receptors detectable with the hydrophilic ligand [3H]-CGP-12177. This loss is attributable to a reduction in both maximal beta-receptor numbers and their affinities. The synthetic diacylglycerol, 1-oleyl-2-acetyldiglycerol, which is known to activate protein kinase C, also induces desensitization of beta-receptors. Both phorbol dibutyrate and 1-oleyl-2-acetyldiglycerol have additive effects to isoproterenol, suggesting a separate site of action in promoting beta-receptor desensitization. The effects of phorbol dibutyrate and 1-oleyl-2-acetyldiglycerol are prevented by colchicine (but not its inactive analog, trimethylcolchicinic acid), indicating a microtubule dependence. The loss of membrane-bound beta-receptors after phorbol dibutyrate- or 1-oleyl-2-acetyldiglycerol preincubation is accompanied by an increase in beta-receptors associated with a cytosol-derived vesicular fraction devoid of plasma membrane markers, a finding consistent with an internalization process. These results suggest that protein kinase C activation by diacylglycerols derived from receptor-linked phosphoinositide hydrolysis may be a novel mechanism of cardiac beta-receptor desensitization.