Bradykinin (BK)-stimulated release of endothelium-derived relaxing factor has been linked to a rise in cytosolic Ca2+ concentration and a change of K+ permeability of the endothelial cell. In the present study, measurement of BK-induced changes in fura-2 fluorescence and 86Rb+ efflux were used to monitor changes in cytosolic Ca2+ and K+ permeability in cultured bovine aortic endothelial cells. In the presence of normal extracellular Ca2+, BK induced a fourfold increase in cytosolic Ca2+, which peaked at 20 s and declined within 1 min to a value that was 50% of the peak level. Subsequently, cytosolic Ca2+ decreased and approached basal levels within 8 min. In the absence of Ca2+, BK produced a 1.5- to 2-fold increase in cytosolic Ca2+ that peaked within 20 s and declined to basal levels within 2 min. Addition of Ca2+ to the Ca-free reaction buffer 3-5 min after addition of BK resulted in a two-to threefold increase in cytosolic Ca2+ that declined slowly back to basal levels. Thus Ca2+ influx can occur in response to BK at a time when there is minimal elevation of cytosolic Ca2+ above the resting level. Under all conditions tested, 86Rb+ efflux paralleled changes in the cytosolic Ca2+, suggesting that efflux occurred through Ca2+-activated K+ channels. Isosmotic substitution of Na+ with N-methyl-D-glucamine did not affect the BK-stimulated changes in cytosolic Ca2+ or 86Rb+ efflux, suggesting that Na+-Ca2+ exchange plays little role in the BK response. These results suggest that BK stimulates Ca2+ influx via a BK receptor-operated channel or a channel activated by some internal messenger other than Ca2+.