Extracellular matrix (ECM) components significantly influence the growth characteristics of cardiomyocytes, development of spontaneous contractile activity and morphologic differentiation. The present study involves characterization of ES cell-derived cardiomyocytes cultured on different ECMs. We hypothesized that cardiogel, which is a naturally occurring ECM containing a complex mixture of laminin, fibronectin may better support ES cell-derived cardiomyocytes maturation in terms of chronotropic characteristics and subcellular structure development. Spontaneously differentiated cardiomyocytes from 7-day embryoid bodies (EBs) derived from mouse ES cells line Royan B1 (C57BL/6) were cultured on commercial ECM (matrigel), a fibroblast-derived ECM (cardiogel), and control (without ECM) for 7 + 21 days. The growth characteristics of cardiomyocytes were assessed by immunocytochemistry, reverse transcription polymerase chain reaction (RT-PCR), positive and negative chronotropic drugs and transmission electron microscopy (TEM). The spontaneously beating cells expressed markers characteristic of cardiomyocytes including alpha-actinin, desmin, troponin-I, sarcomeric myosin heavy chain (MHC), pan-cadherin, connexin-43, cardiac alpha-MHC, cardiac beta-MHC, atrial natriuretic factor (ANF) and myosin light chain isoform-2V (MLC-2V). In addition, spontaneous beating of cardiomyocytes on both ECMs was enhanced by treatment of cells with isopernaline, while reduced more on matrigel, by carbacol when compared with control and cardiogel. However, the change in beating was similar in all groups upon treatment with phenylephrine and/or Bay-K. Ultrastructure analysis showed myofibrillar bundle organization and exhibited intercalated discs, T-tubules, Z-, A-, I-, H- and M-bands and the showed earlier maturation of the cardiomyocytes on cardiogel. We conclude that substructurally matured cardiomyocytes can be produced from ES cells and the cardiac fibroblast-derived ECM (cardiogel) supported earlier maturation of ES-derived cardiomyocytes, in terms of chronotropic characteristics and subcellular structure development.