The incorporation of [32P]Pi into sarcolemmal, sarcoplasmic reticular and myofibrillar proteins was studied in Langendorff-perfused guinea pig hearts treated with the alpha-agonist norepinephrine or with protein kinase C activators (phorbol 12-myristate 13-acetate (PMA) or 1,2-dioctanoylglycerol (D8G]. Norepinephrine was administered in the presence of propranolol and atropine, while the protein kinase C activators (PMA and D8G) were infused in the presence of propranolol, atropine and prazosin. Examination of 32P-incorporation into the various cardiac proteins revealed that there were no significant increases in the degree of phosphorylation of the: (1) 15 kDa sarcolemmal protein; (2) phospholamban in sarcoplasmic reticulum; and (3) troponin I and C protein in the myofibrils. In parallel control studies, stimulation of beating guinea pig hearts by isoproterenol was associated with a 4-5-fold increase in 32P-incorporation into phospholamban and troponin I and about a 2-fold increase in 32P-incorporation into C protein and the 15 kDa sarcolemmal protein. These findings indicate that the major cardiac regulatory phosphoproteins, which have been reported to serve as substrates for protein kinase C in vitro, are not phosphorylated by the same enzyme in perfused, beating guinea pig hearts.