Isolated bovine cerebral microvessels (ICMV) were incubated with different metabolic fuels to determine their ability to support microvessel Na(+)-K(+)-ATPase (quantitated as ouabain-sensitive 86Rb+ uptake) and the ATP/ADP ratio. In comparison with ICMV incubated with glucose, Na(+)-K(+)-ATPase activity was reduced by 55% after a 3-h incubation in fuel-free medium and by 30-40% after incubation with beta-hydroxybutyrate, acetoacetate, or glutamate. However, Na(+)-K(+)-ATPase activity was not significantly decreased in ICMV incubated with pyruvate or oleate plus carnitine. In contrast, only glucose was able to maintain the ATP/ADP ratio. To evaluate the effect of endogenous fatty acid metabolism on these parameters, ICMV were incubated with bromostearate, an inhibitor of fatty acid oxidation. Bromostearate decreased both Na(+)-K(+)-ATPase activity and the ATP/ADP ratio, even in the presence of glucose. These results indicate that the varying effects of different fuels on Na(+)-K(+)-ATPase in ICMV cannot be explained solely by their effects on the ATP/ADP ratio or on glycolytic ATP generation. They suggest that other fuel-modulated factors play a key role in regulating this enzyme.