Insulin can alter myocardial contractility, in part through an effect on the cardiac sarcolemmal Na(+)/Ca(2+) exchanger (NCX), but little is known about its mechanism of action. The large cytoplasmic domain (f-loop) of NCX is required for regulation by various intracellular factors, and we have shown previously that residues 562-679 are determinants of NCX inhibition by exchanger inhibitory peptide (XIP). Here we show that the same f-loop deletion eliminates the enhancement of NCX current by insulin, and we examine the signal pathways involved in the insulin response. NCX current (I(NCX)) was measured in freshly isolated or cultured (up to 48 h) adult guinea pig myocytes and in myocytes expressing canine NCX1.1 with the 562-679 f-loop deletion (NCX-(Delta562-679)) via adenoviral gene transfer. I(NCX) was recorded by whole-cell patch clamp as the Ni(2+)-sensitive current at 37 degrees C with intracellular Ca(2+) buffered. Insulin (1 microm) increased I(NCX) (at +80 mV) by 110 and 83% in fresh and cultured myocytes, respectively, whereas in myocytes expressing NCX-(Delta562-679) the response was eliminated (with 100 microm XIP included to suppress any native guinea pig I(NCX)). The insulin effect on I(NCX) was not inhibited by wortmannin, a nitric-oxide synthase inhibitor, or disruption of caveolae but was blocked by chelerythrine, implicating protein kinase C, but not phosphatidylinositol-3-kinase, in the mechanism. The insulin effect was also not additive with phosphatidylinositol-4,5-bisphosphate-induced activation of I(NCX). The finding that the 562-670 f-loop domain is implicated in both XIP and receptor-mediated modulation of NCX highlights its important role in acute physiological or pathophysiological regulation of Ca(2+) balance in the heart.