Vascular permeability is regulated by endothelial cytosolic Ca²⁺concentration ([Ca²⁺]_i). To determine whether vascular permeability is dependent on extracellular Ca²⁺influx or release of Ca²⁺ from stores, hydraulic conductivity (L _p) was measured in single perfused frog mesenteric microvessels in the presence and absence of Ca²⁺ influx and store depletion. Prevention of Ca²⁺ reuptake into stores by sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) inhibition increasedL _p in the absence of extracellular Ca²⁺ influx. L _p was further increased when Ca²⁺ influx was restored. Depletion of the Ca²⁺ stores with ionomycin and SERCA inhibition increasedL _p in the presence and the absence of extracellular Ca²⁺ influx. However, store depletion in itself did not significantly increase L _p in the absence of active Ca²⁺ release from stores into the cytoplasm. There was a significant positive correlation between baseline permeability and the magnitude of the responses to both Ca²⁺ store release and Ca²⁺ influx, indicating that the Ca²⁺ regulating properties of the endothelial cells may regulate the baseline L _p. To investigate the role of Ca²⁺ stores in regulation ofL _p, the relationship between SERCA inhibition and store release was studied. The magnitude of theL _p increase during SERCA inhibition significantly and inversely correlated with that during store release by Ca²⁺ ionophore, implying that the degree of store depletion regulates the size of the increase onL _p. These data show that microvascular permeability in vivo can be increased by agents that release Ca²⁺ from stores in the absence of Ca²⁺ influx. They also show that capacitative Ca²⁺ entry results in increased L _p and that the size of the permeability increase can be regulated by the degree of Ca²⁺ release.