We investigated the effects of simulated ischemia on intracellular Cl activity ([Cl]_i) in isolated guinea pig ventricular papillary muscles using ion-selective microelectrode techniques. Simulated ischemia in ventricular muscles was produced by stopping the flow of superfusion and immersing preparations in mineral oil as previously described [B. Vanheel, L. Leybaert, A. De Hemptinne, and I. Leusen. Am. J. Physiol. 257 (Cell Physiol. 26): C365-C379, 1989; Z. F. Lai, J. Liu, and K. Nishi. Jpn. J. Pharmacol. 72: 161-174, 1996]. When preparations were exposed to paraffin oil for 15 min, [Cl]_i markedly increased and the peak magnitude of [Cl]_i reached 55.3 ± 2.5 mM from 18.7 ± 3.5 mM, whereas membrane potentials (V_m) depolarized from 82.5 ± 1.1 to 54.7 ± 2.4 mV (n = 6 muscles from 6 animals). SITS (0.5 mM), a known blocker of the Cl/HCO₃ exchanger, suppressed the ischemia-induced depolarization of V_m and delayed the onset of the ischemia-induced increase in [Cl]_i but did not suppress the magnitude of the increase of [Cl]_i. Under Cl-free conditions created by replacing Cl with equimolar gluconate, the increase in [Cl]_i during ischemia was transient and suppressed by >60% compared with that in normal-Cl conditions (peak value was 20.3 ± 1.7 mM, n = 6 muscles from 6 animals). The present results provide direct evidence that [Cl]_i in ventricular muscle increases in ischemic conditions in quiescent guinea pig ventricular muscle, suggesting that activation of the Cl/HCO₃ exchanger by ischemia would partially contribute to the elevation of [Cl]_i during the initial stage of ischemia.