A specific and sensitive detection method for nitric oxide (NO) in living cells and tissue culture systems is required in the search for novel NO synthase (NOS) inhibitors. We have improved a fluorometric determination with 2,3-diaminonaphthalene (DAN) by the addition of 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO) as an oxidant to form NO2 from NO. This method is 3 times more sensitive than that without PTIO, and is suitable for examining the NOS-inhibitory activity of large numbers of test compounds using a 96-well microplate reader. The improved method was applied to N-monomethyl-L-arginine (L-NMMA) as a known inhibitor and the derivatives of 2-phenyl-1,2-benzisoselenazol-3(2H)-one as teat compounds in order to investigate the effect of these compounds on NO production from activated rat aortic smooth muscle cells. The results obtained indicate that this method is suitable for the rapid assay of large numbers of test compounds.