The three isoforms of transforming growth factor-β (TGF-β) have previously been implicated in embryonic development of the heart as well as in repair of myocardial damage after ischemia/reperfusion injury. TGF-β1 has also been localized intracellularly to both mitochondria and contractile filaments of cardiac myocytes, although its role in these structures has not been defined. We now report that exogenous TGF-β stabilizes the beating rate of neonatal rat cardiac myocytes cultured on fibroblast matrix, and sustains their spontaneous rhythmic beating in serum-free medium. Moreover, using blocking antibodies to TGF-β, we show that endogenous TGF-β secreted by these myocytes acts in an autocrine fashion to maintain their beating rate. In contrast, IL-1β, an inflammatory mediator secreted by immune cells during myocardial injury, inhibits the beating of cardiac myocytes, and TGF-β can overcome this inhibition. The antagonistic effects of TGF-β and IL-1 were not observed when the myocytes were cultured on gelatin, as compared to native fibroblast matrix. The data indicate that TGF-β is an important regulator of contractile function of the heart and have significant implications for understanding cardiac physiology in health and disease.