The sodium-calcium exchanger is critical in the normal functioning of many cells. In heart muscle, it is the principal way by which the cells keep the concentration of intracellular calcium low, pumping out the Ca2+ that enters the cytosol through L-type Ca2+ channels. The exchanger may also contribute to the triggering of Ca2+ release during voltage-activated excitation-contraction coupling in heart. Time resolved examination of the conformational changes of macromolecules in living cells has so far been largely restricted to ion-channel proteins whose gating is voltage-dependent. We have now directly measured electrical currents arising from the molecular rearrangements of the sarcolemmal Na-Ca exchanger. Changes in the conformation of the exchanger protein were activated by a rapid increase in the intracellular calcium concentration produced by flash photolysis of caged calcium in voltage-clamped heart cells. Two components of membrane current were produced, reflecting a calcium-dependent conformational change of the transporter proteins and net transport of ions by the exchanger. The properties of these components provide evidence that the Na-Ca exchanger protein undergoes two consecutive membrane-crossing molecular transitions that each move charge, and that there are at least 250 exchangers per micron 2 turning over up to 2,500 times per second.