Effects of the heparin-binding growth factor midkine (MK) were investigated on endothelial Ca2+ signaling and nitric oxide (NO) production. Bradykinin (10 nM) and thapsigargin (1 mu M) provoked large Ca2+ influxes under fura-2/AM fluoroscopy. Pretreatment with human MK dose-dependently (1-500 ng/ml) inhibited the Ca2+ response to bradykinin but not that to thapsigargin. Anti-MK antibody prevented this effect. In Ca2+-free medium, MK greatly inhibited intracellular Ca2+ store release by bradykinin and not that by thapsigargin, which effect was prevented by the antibody. Bradykinin increased NO production by 6.7-fold, which was inhibited 6, 44, 79, and 90% by MH at 1, 10, 100, and 500 ng/ml, respectively. MK did not affect thapsigargin-induced NO production. Our data clearly indicate that MH inhibits bradykinin-induced Ca2+ response and NO production from endothelial cells.