We present the physical and biochemical effects of extracellular matrixes (ECMs) on HL-1 cardiomyocytes. ECMs play major roles in cell growth, adhesion and the maintenance of native cell functions. We investigated the effects of 6 different cell culture systems: 5 different ECM-treated surfaces (fibronectin, laminin, collagen I, gelatin and a gelatin/fibronectin mixture) and 1 nontreated surface. Surface morphology was scanned and analyzed using atomic force microscopy in order to investigate the physical effects of ECMs. The attachment, growth, viability, proliferation and phenotype of the cells were analyzed using phase-contrast microscopy and immunocytochemistry to elucidate the biochemical effects of ECMs. Our study provides basic information for understanding cell-ECM interactions and should be utilized in future cardiac cell research and tissue engineering.