To determine whether age-dependent differences in cardiac responses to autonomic agonists could result from developmental changes in protein kinase C (PKC) isoform expression, we probed extracts from the fetal, neonatal, and adult heart as well as cultured neonatal and isolated adult ventricular myocytes with specific antisera to calcium-dependent (α and β) and calcium-independent (δ, ε, and ζ) isoforms of the enzyme. Although PKC-β immunoreactivity could not be detected in cultured neonatal or isolated adult ventricular myocytes, adult and neonatal myocytes expressed multiple other isoforms of PKC. Our studies revealed an age-dependent decline in the immunoreactivity for three PKC isoforms. PKC-α was detected in extracts from the fetal and 2-day-old neonatal heart as well as cultured neonatal rat ventricular myocytes. Only faint PKC-α immunoreactivity was detected in extracts from the adult heart, and PKC-α was not detected in extracts from isolated adult ventricular myocytes, suggesting that PKC-α resides in nonmyocyte elements in the adult heart. PKC-δ also was detected in greater abundance in fetal and neonatal than in adult myocardial extracts. The decline in PKC-α and PKC-δ expression occurred during the first 2 postnatal weeks. PKC-ζ was detected in greatest abundance in extracts from the fetal heart. PKC-ζ expression declined markedly by the second postnatal day, and only faint PKC-ζ immunoreactivity was detected in extracts from adult myocardium. Failure to detect PKC-ζ in extracts from isolated adult ventricular myocytes suggests that PKC-ζ resides primarily in nonmyocyte elements in the adult heart. PKC-ε was detected in all preparations, but it was detected in greatest abundance in extracts from neonatal hearts. In vitro sympathetic innervation of previously noninnervated neonatal ventricular myocytes or in vivo chemical sympathectomy of the neonatal heart did not modulate PKC isoform expression, suggesting that sympathetic innervation does not significantly regulate PKC isoform expression. PKC-α partitioned to the soluble fraction of unstimulated myocytes and was selectively translocated to the particulate fraction by Ca2+. In contrast, a major portion of the novel PKC isoforms partitioned to the particulate fraction of unstimulated myocytes. The subcellular distribution of novel PKC isoforms was not influenced by Ca2+. 12-O-Tetra-decanoylphorbol 13-acetate (TPA, 300 nmol/L) induced trans-location of soluble PKC-α, PKC-δ, and PKC-ε to the particulate fraction at 30 minutes and complete (PKC-α and PKC-δ) or 80% (PKC-ε) downregulation at 24 hours. PKC-ζ was not affected by TPA. We conclude that multiple PKC isoforms, which differ in their subcellular distribution and regulation by Ca2+ and phorbol esters, are expressed in the heart in an age-dependent fashion. The observation that the developmental decline in PKC-ζ precedes the fall in PKC-α and PKC-δ suggests that PKC isoform expression is controlled by distinct mechanisms that are regulated differently during development.