Background - Cardiac memory refers to an altered T-wave morphology induced by ventricular pacing or arrhythmias that persist for variable intervals after resumption of sinus rhythm. Methods and Results - We induced long-term cardiac memory (LTM) in conscious dogs by pacing the ventricles at 120 bpm for 3 weeks. ECGs were recorded daily for 1 hour, during which time pacing was discontinued. At terminal study, the heart was removed and the electrophysiology of left ventricular epicardial myocytes was investigated. Control (C) and LTM ECG did not differ, except for T-wave amplitude, which decreased from 0.12 ± 0.18 to -0.34 ± 0.2] mV (±SEM, P<0.05), and T-wave vector, which shifted from -37 ± 12°to -143 ± 4°(P<0.05). Epicardial action potentials revealed loss of the notch and lengthening of duration at 20 days (both P<0.05). Calcium-insensitive transient outward current (I(to)) was investigated by whole-cell patch clamp. No difference in capacitance was seen in C and LTM myocytes. I(to) activated on membrane depolarization to - 25 ± 1 mV in C and -7 ± 1 mV (P<0.05) in LTM myocytes, indicating a positive voltage shift of activation. I(to) density was reduced in LTM myocytes, and a decreased mRNA level for Kv4.3 was observed. Recovery of I(to) from inactivation was significantly prolonged: it was 531 ± 80 ms (n=10) in LTM and 27 ± 6 ms (n=9) in C (P<0.05) at -65 mV. Conclusions - I(to) changes are associated with and can provide at least a partial explanation for action-potential and T-wave changes occurring with LTM.