Inward rectification, an important determinant of cell excitability, can result from channel blockade by intracellular cations, including Ca2+. However, mostly on the basis of indirect arguments, Ca2+-mediated rectification of inward rectifier K+ current (IK1) is claimed to play no role in the mammalian heart. The present study investigates Ca2+-mediated IK1 rectification during the mammalian ventricular action potential. Guinea pig ventricular myocytes were patch-clamped in the whole-cell configuration. The action potential waveform was recorded and then applied to reproduce normal excitation under voltage-clamp conditions. Subtraction currents obtained during blockade of K+ currents by either 1 mmol/L Ba2+ (IBa) or K+-free solution (I0K) were used to estimate IK1. Similar time courses were observed for IBa and I0K; both currents were strongly reduced during depolarization (inward rectification). Blockade of L-type Ca2+ current by dihydropyridines (DHPs) increased systolic IBa and I0K by 50.7% and 254.5%, respectively. beta-Adrenergic stimulation, when tested on I0K, had an opposite effect; ie, it reduced this current by 66.5%. Ryanodine, an inhibitor of sarcoplasmic Ca2+ release, increased systolic IBa by 47.7%, with effects similar to those of DHPs. Intracellular Ca2+ buffering (BAPTA-AM) increased systolic IBa by 87.7% and blunted the effect of DHPs. Thus, IK1 may be significantly reduced by physiological Ca2+ transients determined by both Ca2+ influx and release. Although Ca2+-induced effects may represent only a small fraction of total IK1 rectification, they are large enough to affect excitability and repolarization. They may also contribute to facilitation of early afterdepolarizations by conditions increasing Ca2+ influx.