We have developed diaminofluoresceins (DAFs) and diaminorhodamines as fluorescent indicators for NO based on the specific reaction of the aromatic vicinal diamines with NO. Among them, 4,5-diaminofluorescein (DAF-2) is widely used for real-time biological imaging of NO in cultured cells or tissues by many researchers. Contrary to a recent report of divalent cation sensitivity and photoactivation of DAF-2 (Broillet, M. C., Randin, O., and Chatton, J. Y. (2001) FEBS Lett. 491, 227-232), our study using NO gas itself reveals that the reaction of DAF-2 and NO is completely independent of Ca2+ and Mg2+ at physiological concentrations. Ca2+ enhances not the conversion of DAF-2 into its fluorescent product (DAF-2 triazole) but the release of NO from NO donors. Therefore it is concluded that DAF-2 can provide reliable information on NO production in biological systems regardless of the dynamic changes of Ca2+ concentration.