We investigated the localization of the Na-Ca exchanger in fixed, isolated heart cells from rat and guinea pig using immunocytochemical methods with epifluorescence and confocal microscopy. We found that the Na-Ca exchanger is distributed throughout all membranes in contact with the extracellular space, including the sarcolemma, the transverse tubules (T-tubules), and the intercalated disks. Microscopic nonuniformities in the fluorescent labeling appear to reflect varying views of the membranes containing Na-Ca exchanger protein. Confocal thin-section imaging reveals a regular grid of discrete foci of fluorescence, which represent Na-Ca exchanger in T-tubules viewed en face. These foci are 1.80 +/- 0.01 microns apart from sarcomere to sarcomere and are aligned with the Z-line. Along each Z-line, these foci are spaced at 1.22 +/- 0.11-microns intervals. Longitudinal sections of the sarcolemma-T-tubule junction show a comblike appearance, with T-tubules extending inward from the heavily labeled sarcolemma. Our finding that the Na-Ca exchanger is widely distributed over the cell surface may provide further insight into the role of Na-Ca exchange in the heart.