Isolated preparations of rabbit interventricular septum were perfused through the coronary arteries with oxygenated Tyrode's solution and placed in a tissue bath where they were superfused as well. Transmembrane potentials were simultaneously recorded from the subendocardium with two flexibly mounted microelectrodes, one from a superficial cell, and the other from a deep cell. Ischemia was produced by stopping coronary flow while superfusion with oxygenated Tyrode's solution was maintained. After a 7 to 12 min ischemic period, the preparation was fixed by coronary perfusion with fixative while the microelectrodes remained in place. After fixation, the microelectrodes were withdrawn. Appropriate tissue blocks were cut in 4, gm serial sections and the microelectrode track was followed until the tip position was identified. Transmembrane potentials during ischemia were divided into two categories: (1) “border zone” potentials (resting membrane potential [RMP] 73 + 3 mV, action potential amplitude [APA] 81 + 13 mV, action potential duration [APD] 116 + 48 msec, n = 12) and (2) “ischemic” potentials (RMP 53 ± 4 mV, APA 44 ± 11 mV, APD 102 ± 42 msec, n = 8). Ischemic potentials were recorded from cells at depths greater than 560 pum below the endocardial surface and border zone potentials were recorded in a layer at between 130 and 650 μm below the surface. In a separate series of experiments, extracellular concentrations of K+ and pH were measured with ion-sensitive electrodes at different depths and, after a 10 min period of ischemia, part of the septum was placed in liquid nitrogen to allow determination of phosphocreatine (PC) levels in successive 50 to 100, um layers. After 10 min of ischemia, extracellular K+ gradually increased from 4 to 9 mM in endocardium to a depth of 600 gim, pH fell from 7.4 to 6.6 over the same distance, and PC decreased to very low, stable levels at only 800 gim. It is concluded that in the first 10 min of acute ischemia, an endocardial border zone exists of 40 to 60 cell layers in which transmeimbrane potentials are affected relatively little by ischemia. Within this electrophysiologic border zone extracellular K+ was lower than 9 mM, pH was higher than 6.6, and tissue content of PC was not lower than 40% of normal. In layers deeper than 600 gim, with further development of a metabolic gradient, action potentials became markedly depressed. This electrophysiologic inhomogeneity within the ischemic subendocardium could be a factor in arrhythmogenesis during the first minutes of ischemia.