Using 4,5-diaminofluorescein diacetate (DAF-2DA), which was recently developed for the detection of intracellular nitric oxide (NO) in living cells, we examined the sensitivity of intracellular NO in cells treated with some fixatives. Cultured human umbilical vein endothelial cells loaded with DAF-2DA in the presence of 10^–6 M acetylcholine showed intense fluorescence when fixed in paraformaldehyde or glutaraldehyde, but no fluorescence could be detected after fixation in ethanol or acetone. Fluorescence generation depended on the combination of each aldehyde fixative with DAF-2, which is produced enzymatically from DAF-2DA within the cells. Subtracting the fluorescence intensity of non-activated controls from that of cells activated by acetylcholine indicated the NO produced in the stimulated cells, since the control cells that took up DAF-2DA also generated fluorescence when treated with aldehyde fixatives. Thus, detection of intracellular NO by combining aldehyde fixatives with DAF-2DA is useful for examining the functions of NO in cells both in situ and in vivo.