Vasostatin-1 (VS-1), the N-terminal fragment derived from the cleavage of chromogranin A (CgA), has been shown to exert several biological activities on several tissues and organs. Recently, it has been reported that human recombinant VS-1 (STA-CGA1-78) may alter myocardial contractility in eel, frog, and rat hearts. In this article we have explored if STA-CGA 1-78 can induce intracellular cascades interacting both with adhesion molecules and/or extracellular matrix (ECM), components, that is, involvement of the heat shock protein 90 (HSP90) and the endothelial NOS (eNOS), known to be implicated in signal transduction mechanisms affectingmyocardial contractility. We used 3D cultured adult rat cardiomyocytes cultivated over fibronectin or fibroblasts or embedded in matrigel or collagen type I. Aurion-conjugated VS-1 (Au-STA-CGA178) has been used to identify possible sites of interaction of this molecule with the cell membrane. We found that in our 3D culture, cell-ECM interactions played a crucial role in the cellular localization of HSP90 as well as in the expression of eNOS. VS-1 appeared to modulate cell-ECM interactions, thereby remarkably leading to a different cellular localization of HSP90. Moreover, Au-STA-CGA1-78 was never detected inside the cell nor overlapping the plasma membrane, but nearby the outer side of the cardiomyocyte plasmalemma, at a particular distance, typical of integrins. On the whole, these data suggest that VS-1 does not have a classic receptor on the membrane but that integrins may represent a nonconventional VS-1 receptor modulating eNOS signaling pathway.