Graphene is a one-atom thick planar sheet of carbon atoms arranged in a hexagonal crystal lattice. Graphene and related nanomaterials provide a unique biocompatible substrate for the growth of primary and immortalized cardiomyocytes, as well as for the differentiation and maturation of embryonic stem cells into cardiomyocytes. However, the effect of graphene on cardiac ion channels has not been described. The goal of the present study was to determine if culturing cardiomyocytes on graphene alters the properties of cardiac Ca2+ channels. For this purpose immortalized H9c2 rat cardiomyocytes were plated either on uncoated glass coverslips or coverslips coated with a monolayer of graphene synthesized with chemical vapor deposition. Cardiomyocytes plated directly on graphene showed no significant change in the density or current versus voltage relationship of the whole-cell Ba2+ current (IBa) when compared with cells cultured on glass. There was also no difference in the response of IBa in the cardiomyocytes to protein kinase A stimulation or to the dihydropyridines BayK 8644 and nisoldipine. Surprisingly, the H9c2 cells grown on graphene displayed a large reduction in the voltage-dependent inactivation of the Ca2+ channels. Despite this change, there was no alteration in vasopressin-induced Ca2+ transients in the graphene-plated cells. Plating the myocytes on poly-l-lysine- and collagen-coated graphene restored normal inactivation properties to the Ca2+ currents. It is concluded that the unique structural and electrical features of graphene alter the biophysical properties of the Ca2+ channels.