Although cytosolic Ca²⁺ importantly regulates organ function, lung microvascular [Ca²⁺]_i regulation remains poorly understood because of the lack of direct in situ quantification. In the present study, we report the first endothelial [Ca²⁺]_i quantification by the fura 2 method in microscopically imaged venular capillaries of the isolated blood-perfused rat lung. Sequential images indicated the presence of intercellular Ca²⁺ waves that spontaneously originated from pacemaker endothelial cells and then spread for short distances along the capillary wall, inducing synchronous endothelial [Ca²⁺]_i oscillations. Fast Fourier analyses of the oscillations revealed a dominant wave component with an amplitude of 37 nmol/L, frequency of 0.4 min⁻¹, and velocity of 5 μm/s. The intracellular Ca²⁺ wave was unaffected by blood flow stoppage or by infusions of Ca²⁺-containing or Ca²⁺-free dextran. Inhibition of the wave by thapsigargin in Ca²⁺-free dextran and by the gap junction uncoupler, heptanol, indicated that it was generated by endosomal Ca²⁺ release in the pacemaker cell and was propagated by gap junctional communication. In the presence of histamine, enhancement of the wave accounted for a significant component of the coordinated [Ca²⁺]_i increase in the capillary segment. No intercellular Ca²⁺ waves were evident in adjoining alveolar epithelial cells. Our findings indicate a novel mechanism of [Ca²⁺]_i regulation in the lung capillary under both resting and stimulated conditions. Pacemaker-induced Ca²⁺ waves, generated intracellularly by unknown initiating mechanisms, communicated to adjoining cells to determine [Ca²⁺]_i profiles in short interbranch segments of capillary walls.