The catecholamine release inhibitory catestatin (Cts; human CgA_352-372, bovine CgA_344-364) is a vasoreactive and hypertensive peptide derived from Chromogranin A (CgA). Using the isolated avascular frog heart as a bioassay, in which the interactions between the endocardial endothelium (EE) and the subjacent myocardium can be studied without the confounding effects of the vascular endothelium, we tested the direct cardiotropic effects of bovine catestatin and its interaction with -adrenergic (isoproterenol, ISO) and endothelin-1 (ET-1) signaling. Cts dose-dependently decreased stroke volume and stroke work, with a threshold concentration of 11 nM, approaching the in vivo level of the peptide. Cts reduced contractility by inhibiting phosphorylation of phospholamban. Furthermore, the Cts effect was abolished by pre-treatment with either Nitric Oxide Synthase (L-NMMA) or cGMP inhibitors (ODQ), or an ET_B receptor (ET_BR) antagonist (BQ788). Cts also non-competitively inhibited the positive inotropic action of ISO. In addition, Cts inhibited the positive inotropic effect of ET-1, mediated by ET_A receptors (ET_AR), and did not alter the negative-inotropic ET-1 influence mediated by ET_BR. Cts action through ET_BR was further suggested when, in the presence of BQ788, Cts failed to inhibit the positive inotropism of both ISO and ET-1 stimulation and phospholamban (PLN) phosphorylation. We conclude that the cardiotropic actions of Cts, including the -adrenergic and ET-1 antagonistic effects, support a novel role of this peptide as an autocrine-paracrine modulator of cardiac function, particularly when the stressed heart becomes preferential target of both adrenergic and endothelin stimuli.