Our novel fluorescent indicator, DAF-FM, permits the bioimaging of nitric oxide (NO) in living cells with high resolution in space and time, with stable intensity above pH 5.8. A membrane-permeable derivative, DAF-FM DA, was applied to imaging of NO generated in rat hippocampal slices by exposure to an aglycemic medium. NO production was observed mainly in the CA1 area, and was dependent on the concentration of O(2). During exposure to an anoxic-aglycemic medium, NO was hardly produced, while marked elevation of intracellular Ca(2+) was observed. Production of NO increased sharply as soon as the perfusate was changed to the normal medium. These results suggest that NO synthase is activated after reperfusion rather than during ischemia.