It is often assumed that the global cytosolic Ca²⁺ concentration ([Ca²⁺]_c) is responsible for setting microvascular permeability. We have previously shown that, on average, agonist induced permeability responses reach a maximum before the [Ca²⁺]_c reaches a maximum (Glass et al, JVR, 30:289, 2003) suggesting that the changes in permeability precede those in Ca²⁺ and are therefore not caused by them. In this study we aimed to investigate the effect of reducing [Ca²⁺]_c on basal permeability. The Landis-Michel method (Michel et al, QJEPCMS, 1974;59:283) was used to measure hydraulic conductivity (permeability to water, L_p) (x10^-7 cm.s^-1.cmH₂O^-1) in single mesenteric microvessels of frog. Erythrocytes were collected by cardiac puncture from 5% halothane anaesthetised rats (killed by cervical dislocation). Frogs were anaesthetised by immersion in 1mg.ml^-1 MS222 in frog Ringer, and anaesthesia was maintained by superfusing the mesentery with 0.25mg.ml^-1 MS222. Frogs were humanely killed by cranial destruction at the end of the experiment. All experiments conformed with the national guidelines for the usage of animals. Microvessels were cannulated and perfused with 1% bovine serum albumin (BSA) and the basal L_p was measured. Then the vessels were perfused with 25μM BAPTA-AM (O,O’-Bis(2-aminophenyl) ethyleneglycol-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester ) in 1% BSA for 10 min to chelate the cytosolic Ca²⁺ and L_p was measured a second time. Basal L_p (3.3±0.7, median±interquartile range) during perfusion with BSA was significantly reduced to 0.8±0.1 following 10 min perfusion with BAPTA (p<0.02, n=7, Wilcoxon). These results alone could be used to suggest that basal permeability is regulated by the [Ca²⁺]_c. To confirm that the [Ca²⁺]_c was reduced during the 10 min treatment with BAPTA we measured the [Ca²⁺]_c in Fura-2 loaded microvessels before and after the addition of 25μM BAPTA using the protocol above. Basal [Ca²⁺]_c, represented by a normalised ratio (R_norm) of the emission fluorescence at excitation wavelengths of 340nm and 380nm of. The R_norm decreased slightly, but not significantly, from 10.7±4.2 (mean±sem) to an R_norm of 7.6±2.3 following 10 min exposure to BAPTA (p>0.05, n=6, paired t-test). As a whole, these experiments suggest that basal permeability is not set by the global endothelial cell [Ca²⁺]_c, as perfusion with BAPTA significantly reduces L_p without affecting [Ca²⁺]_c. Therefore, these results support the hypothesis that basal permeability is regulated by the [Ca²⁺] in microdomains within the cell, such as the in the ER or around the ER Ca²⁺ leak channels.