Endogenous nitric oxide (NO) appears to modulate many physiological and pathophysiological processes. In order to obtain direct evidence for NO functions in vivo, we have developed 4,5-diaminofluorescein (DAF-2) as a novel fluorescent indicator for NO. Green-fluorescent triazolofluorescein formed by the reaction of NO and DAF-2 affords high sensitivity for NO (detection limit: 5 nM). Membrane-permeable DAF-2 diacetate (DAF-2 DA) was loaded into activated rat aortic smooth muscle cells, where the ester bonds are hydrolyzed by intracellular esterase, generating DAF-2. The fluorescence in the cells increased in a NO concentration-dependent manner. This imaging method should be useful for studies of the dynamic biological actions of NO at the molecular level with fine temporal and spatial resolution.