The extracellular matrix (ECM) is a dynamic component of tissues that influences cellular phenotype and behavior. We sought to determine the role of specific ECM substrates in the regulation of protein kinase C (PKC) isozyme expression and function in cardiac myocyte attachment, cell volume, and myofibril formation. PKC isozyme expression was ECM substrate specific. Increasing concentrations of the PKC δ inhibitor rottlerin attenuated myocyte attachment to randomly organized collagen (1, 5, and 10 μM), laminin (5 and 10 μM), aligned collagen (5 and 10 μM), and fibronectin (10 μM). Rottlerin significantly decreased cell volume on laminin and randomly organized collagen, and inhibited myofibril formation on laminin. The PKC α inhibitor Gö 6976 inhibited attachment to randomly organized collagen at 6 nM but did not affect cell volume. The general PKC inhibitor Bisindolylmalemide I (10 and 30 μM) did not affect myocyte attachment; however, it significantly decreased cell volume on randomly organized collagen. Our data indicate that PKC isozymes are expressed and utilized by neonatal cardiac myocytes during attachment, cell growth, and myofibril formation. Specifically, it appears that PKC δ and/or its downstream effectors play an important role in the interaction between cardiac myocytes and laminin, providing further evidence that the ECM influences cardiac myocyte behavior.