Although each of the fundamental processes involved in excitation-contraction coupling in mammalian heart has been identified, many quantitative details remain unclear. The initial goal of our experiments was to measure both the transmembrane Ca²⁺ current, which triggers contraction, and the Ca²⁺ extrusion due to Na⁺-Ca²⁺ exchange in a single ventricular myocyte. An action potential waveform was used as the command for the voltage-clamp circuit, and the membrane potential, membrane current, [Ca²⁺]_i, and contraction (unloaded cell shortening) were monitored simultaneously. Ca²⁺-dependent membrane current during an action potential consists of two components: (1) Ca²⁺ influx through L-type Ca²⁺ channels (I_Ca-L) during the plateau of the action potential and (2) a slow inward tail current that develops during repolarization negative to ≈−25 mV and continues during diastole. This slow inward tail current can be abolished completely by replacement of extracellular Na⁺ with Li⁺, suggesting that it is due to electrogenic Na⁺-Ca²⁺ exchange. In agreement with this, the net charge movement corresponding to the inward component of the Ca²⁺-dependent current (I_Ca-L) was approximately twice that during the slow inward tail current, a finding that is predicted by a scheme in which the Ca²⁺ that enters during I_Ca is extruded during diastole by a 3 Na⁺–1 Ca²⁺ electrogenic exchanger. Action potential duration is known to be a significant inotropic variable, but the quantitative relation between changes in Ca²⁺ current, action potential duration, and developed tension has not been described in a single myocyte. We used the action potential voltage-clamp technique on ventricular myocytes loaded with indo 1 or rhod 2, both Ca²⁺ indicators, to study the relation between action potential duration, I_Ca-L, and cell shortening (inotropic effect). A rapid change from a “short” to a “long” action potential command waveform resulted in an immediate decrease in peak I_Ca-L and a marked slowing of its decline (inactivation). Prolongation of the action potential also resulted in slowly developing increases in the magnitude of Ca²⁺ transients (145±2%) and unloaded cell shortening (4.0±0.4 to 7.6±0.4 μm). The time-dependent nature of these effects suggests that a change in Ca²⁺ content (loading) of the sarcoplasmic reticulum is responsible. Measurement of [Ca²⁺]_i by use of rhod 2 showed that changes in the rate of rise of the [Ca²⁺]_i transient (which in rat ventricle is due to the rate of Ca²⁺ release from the sarcoplasmic reticulum) were closely correlated with changes in the magnitude and the time course of I_Ca-L. These findings demonstrate that Ca²⁺ release from the sarcoplasmic reticulum can be modulated by the action potential waveform as a result of changes in I_Ca-L.