Ghrelin and its synthetic analogue hexarelin are specific ligands of GH secretagogue receptor (GHS-R) and induce a variety of cardiovascular protective and cardiac positive inotropic effects. The signaling system underlying immediate effects of both GHSs in cardiomyocytes remains undefined. In the present study, we investigated the immediate effects of GHSs on isolated ventricular myocyte shortening, intracellular Ca2+ ([Ca 2+]i) transients, and the L-type Ca2+ current (ICa,L). Putative intracellular signalling cascades were studied with specific receptor and signalling blockers. In fresh isolated adult Wistar rat ventricular myocytes, GHSs produced a positive inotropic effect in a concentration-dependent manner and increased the amplitude of [Ca 2+]i transients and the ICa,L. The positive inotropic response was abolished by the GHS-R1a antagonist [D-Lys 3]-GH-releasing peptide-6 (10 μM). GHS-induced increase in the ICa,L was abolished by [D-Lys3]-GH-releasing peptide-6 and protein kinase C inhibitor, chelerythrine chloride (5 μM), but not by protein kinase A inhibitor, KT 5720 (10 μM). We conclude that hexarelin and ghrelin increase the ICa,L, through GHS-R1a receptor and protein kinase C signalling cascade, which contribute to its direct positive inotropic effect on cardiomyocytes. Copyright © 2010 by The Endocrine Society.