The influence of lack of oxygen and metabolic inhibitors on the integrity of cerebral microvascular endothelium is undefined due to conflicting experimental evidence. A newly developed technique for continuous measurement of the electrical resistance of the microvascular endothelium was applied to the pial venules of the frog subjected to severe hypoxia and inhibition of endothelial ATP-production by means of cyanide (1 mM) and iodo-acetate (1 mM) for periods of 15 min. Severe hypoxia as well as application of the metabolic inhibitors decreased the electrical resistance within 2-5 min. The effect of the glycolysis-blocking agent, iodo-acetate, was particularly powerful. The maximal response, obtained in a 15 min period of exposure, was a 30-50% resistance decrease, corresponding to a 50-100% increase in the permeability to small ions. The rapid rise in permeability demonstrates that the cerebral endothelial cells depend critically on uninterrupted energy conversion to maintain the barrier function.