Excitation-contraction coupling was studied in mammalian cardiac cells in which the opening probability of L-type calcium (Ca2+) channels was reduced. Confocal microscopy during voltage-clamp depolarization revealed distinct local transients in the concentration of intracellular calcium ions ([Ca2+](i)). When voltage was varied, the latency to occurrence and the relative probability of occurrence of local [Ca2+](i) transients varied as predicted if Ca2+ release from the sarcoplasmic reticulum (SR) was linked tightly to Ca2+ flux through L-type Ca2+ channels but not to that through the Na-Ca exchanger or to average [Ca2+](i). Voltage had no effect on the amplitude of local [Ca2+](i) transients. Thus, the most efficacious "Ca2+ signal" for activating Ca2+ release from the SR may be a transient microdomain of high [Ca2+](i) beneath an individual, open L-type Ca2+ channel.