-Glutamyl transpeptidase (-GT) is a key enzyme in GSH metabolism that regulates intracellular GSH levels in response to extracellular GSH (GSH_o). The objective of this study was to identify the role of -GT in reversing pathogenic K⁺ channel remodeling in the diseased heart. Chronic ventricular dysfunction was induced in rats by myocardial infarction (MI) and studies were done after 6-8wks. Biochemical assays of tissue extracts from post-MI hearts revealed significant increases in -GT activity in left ventricle (47%) and septum (28%) compared with sham hearts, which paralleled increases in protein abundance and mRNA. Voltage-clamp studies of isolated left ventricular myocytes from post-MI hearts showed that down-regulation of transient outward K⁺ current (I_to) was reversed after 4-5 h by 10 mmol/l GSH_o or N-acetylcysteine (NAC_o), and that the effect of GSH_o but not NAC_o was blocked by the -GT inhibitors, acivicin or S-hexyl-GSH. Inhibition of -glutamylcysteine synthetase by buthionine sulfoximine did not prevent up-regulation of I_to by GSH_o, suggesting that intracellular synthesis of GSH was not directly involved. However, pre-treatment of post-MI myocytes with an SOD mimetic (MnTPyP) and catalase completely blocked recovery of I_to by GSH_o. Confocal microscopy using the fluorogenic dye 2', 7' -dichlorodihydrofluorescein diacetate confirmed that GSH_o increased reactive oxygen species (ROS) generation by post-MI myocytes and to a lesser extent in myocytes from sham hearts. Furthermore, GSH_o-mediated up-regulation of I_to was blocked by inhibitors of tyrosine kinase (genistein, lavendustin A, AG1024) and thioredoxin reductase (auranofin, 13-cis-retinoic acid). These data suggest that GSH_o elicits -GT- and ROS-dependent transactivation of tyrosine kinase signaling that up-regulates K⁺ channel activity or expression via redox-mediated mechanisms. The signaling events stimulated by -GT catalysis of GSH_o may be a therapeutic target to reverse pathogenic electrical remodeling of the failing heart.