Chromogranin A (CGA), produced by human and rat myocardium, generates several biologically active peptides processed at specific proteolytic cleavage sites. A highly conserved cleavage N-terminal site is the bond 64–65 that reproduces the native rat CGA sequence (rCGA1–64), corresponding to human N-terminal CGA-derived vasostatin-1. rCGA1–64 cardiotropic activity has been explored in rat cardiac preparations. In Langendorff perfused rat heart, rCGA1–64 (from 33 nM) induced negative inotropism and lusitropism as well as coronary dilation, counteracting isoproterenol (Iso) - and endothelin-1 (ET-1) -induced positive inotropic effects and ET-1-dependent coronary constriction. rCGA1–64 also depressed basal and Iso-induced contractility on rat papillary muscles, without affecting calcium transients on isolated ventricular cells. Structure-function analysis using three modified peptides on both rat heart and papillary muscles revealed the disulfide bridge requirement for the cardiotropic action. A decline in Iso intrinsic activity in the presence of the peptides indicates a noncompetitive antagonistic action. Experiments on rat isolated cardiomyocytes and bovine aortic endothelial cells indicate that the negative inotropism observed in rat papillary muscle is probably due to an endothelial phosphatidylinositol 3-kinase-dependent nitric oxide release, rather than to a direct action on cardiomyocytes. Taken together, our data strongly suggest that in the rat heart the homologous rCGA1–64 fragment exerts an autocrine/paracrine modulation of myocardial and coronary performance acting as stabilizer against intense excitatory stimuli.—Cerra, M. C., Gallo, M. P., Angelone, T., Quintieri, A. M., Pulerà, E., Filice, E., Guérold, B., Shooshtarizadeh, P., Levi, R., Ramella, R., Brero, A., Boero, O., Metz-Boutigue, M. H., Tota, B., Alloatti, G. The homologous rat chromogranin A1–64 (rCGA1–64) modulates myocardial and coronary function in rat heart to counteract adrenergic stimulation indirectly via endothelium-derived nitric oxide.