The effect of carbachol (CCh) on the Na/Ca exchange current (I_Na/Ca) was studied in voltage-clamped ventricular myocytes isolated from guinea pig hearts and superfused with Tyrode solution at 35°C. CCh (100 µM) increased outward current during depolarizations (10-200 ms) from 45 mV and tail current amplitude on repolarization; CCh had no effect on the L-type Ca²⁺ current. Amplitudes of the outward and tail currents declined with increasing duration of the depolarizing clamp pulse. Ouabain produced similar current changes that are suppressed by intrapipette ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid and are characteristic of I_Na/Ca. Depolarization from 80 to 30 mV elicited the rapid Na⁺ current followed by a slowly decaying inward I_Na/Ca (J. C. Gilbert, T. Shirayama, and A. J. Pappano. Circ. Res. 69: 1632-1639, 1991.) that was reversibly increased by CCh. Atropine (1-3 µM) prevented the CCh effect. All procedures that suppressed I_Na/Ca also suppressed the CCh effect. Sarcoplasmic reticulum (SR) Ca²⁺ release participated in generating I_Na/Ca because 10 mM caffeine or 1 µM ryanodine blocked I_Na/Ca and the effect of CCh. Rapid superfusion of 10 mM caffeine induced inward I_Na/Ca at 75 mV; a caffeine-induced charge transfer gives an SR Ca²⁺ content of 67 µM. CCh increased caffeine-induced current; SR Ca²⁺ content rose to 98 µM. CCh also augmented the amplitude of steady-state intracellular Ca²⁺ transients and contractions during a train of voltage-clamp pulses (75 to 30 mV for 200 ms) at 1 Hz. CCh elevated intracellular Na⁺ (M. Korth and V. Kühlkamp. Pflügers Arch. 403: 266-272, 1985) by inducing a background Na⁺ current [K. Matsumoto and A. J. Pappano. J. Physiol. (Lond.) 415: 487-502, 1989]. Together with these data, the present results are consistent with the hypothesis that CCh, via muscarinic receptors, eventually promotes I_Na/Ca at the sarcolemma through a mechanism that requires the SR and that this action accounts for the increased contractions.