The protein thiol-modifying agent arsenite, a potent activator of stress signaling, was used to examine the involvement of MAPK's in the regulation of cardiac substrate uptake. Arsenite strongly induced p38 MAPK phosphorylation in isolated rat cardiac myocytes, but also moderately enhanced phosphorylation of p42/44 ERK and p70 S6K. At the level of cardiomyocytic substrate utilization, arsenite enhanced glucose uptake dose-dependently up to 5.1-fold, but failed to stimulate long-chain fatty acid (LCFA) uptake. At the substrate transporter level, arsenite stimulated the translocation of GLUT4 to the sarcolemma but failed to recruit CD36 or FABPpm. Because arsenite did not influence the intrinsic activity of glucose transporters, GLUT4 translocation is entirely responsible for the selective increase in glucose uptake by arsenite. Moreover, the non-additivity of arsenite-induced glucose uptake and insulin-induced glucose uptake indicates that arsenite recruits GLUT4 from insulin-responsive intracellular stores. Inhibitor studies with SB203580/SB202190, PD98059 and rapamycin indicate that activation of p38 MAPK, p42/44 ERK and p70 S6K, respectively, are not involved in arsenite-induced glucose uptake. In addition, all these kinases do not play a role in regulation of cardiac glucose and LCFA uptake by insulin. Hence, arsenite's selective stimulation of glucose uptake appears unrelated to its signaling actions, suggesting that arsenite acts via thiol-modification of a putative intracellular protein target of arsenite within insulin-responsive GLUT4-containing stores. Because of arsenite's selective stimulation of cardiac glucose uptake, identification of this putative target of arsenite within the GLUT4-storage compartment may indicate whether it is a target for future strategies in prevention of diabetic cardiomyopathy.