The extra-cellular matrix has been demonstrated to play an important role in the differentiation of a number of cell types in vitro. The purpose of this study was to establish the role of ECM collagen synthesis in regulating growth and differentiation of embryonic cardiocytes in vitro. We report that treatment of embryonic cardiocytes in vitro with two chemically distinct inhibitors of collagen synthesis, cis-hydroxy-L-proline and ethyl-3,4-dihydroxybenzoate, effectively inhibits collagen secretion. This results in disruption of myofibrillogenesis as seen by immunocytochemistry and electron microscopy, and absence of beating. The expression of muscle specific genes TroponinT and Myosin Heavy Chain are reduced, while the expression of the housekeeping gene glyceraldehyde phosphate dehydrogenase is uneffected. All of these effects are reversible. The structural effects are not prevented by growing the cells on various substrates, including denatured collagen, collagen type IV, laminin and Matrigel. Thus, inhibition of collagen secretion disrupts myofibrillogenesis and results in the alteration of expression of muscle-specific genes, suggesting that collagen synthesis plays an essential role in maintaining the differentiated phenotype of cardiocytes.