Spatial electrical heterogeneity has a profound effect on normal cardiac electrophysiology and genesis of cardiac arrhythmias in diseased hearts. The Na+-Ca2+ exchanger (NCX) is a key linker, through Ca2+ signaling, between contractility and arrhythmias. Here we characterize the differential transmural expression of NCX in normal and rapid pacing-induced failing canine hearts. Significant transmural heterogeneity of NCX was present in normal hearts, as NCX current density measured at +80 mV was significantly (P<0.05) greater in epicardial (EPI) (5.49 pA/pF) than mid-myocardial (MID) (2.84 pA/pF) and endocardial (ENDO) (2.21 pA/pF) cells. Interestingly, heart failure caused a selective increase in NCX current density (P<0.05) limited to ENDO (by 202%) and MID (by 76%) but not EPI myocytes (P=not significant). The differences in functional expression were associated with changes in both mRNA and protein levels. The normal EPI layer exhibited the greatest NCX mRNA and protein levels compared with MID and ENDO layers, whereas the ENDO layer underwent the most pronounced increase in mRNA (by 185%) and protein (by 207%) levels in heart failure. The transmural NCX gradient, from EPI (greatest) to ENDO (least), is disrupted in heart failure. A selective upregulation of NCX expression in MID and ENDO in heart failure markedly redirects the orientation of the transmural functional gradient of NCX and may lead to enhanced vulnerability to cardiac arrhythmias.