A method for the spectrophotometric determination of nitric oxide, nitrite, and nitrate in tissue culture media is presented. The method is based on the nitric oxide-mediated nitrosative modification of sulfanilic acid that reacts with N-(1-naphthyl)ethylenediamine dihydrochloride forming an orange-colored product absorbing at 496 nm. Nitric oxide levels were determined in culture media from this absorbance measurement using chemiluminescence standardization. Extinction coefficients of 5400 and 6600 M - 1 cm - 1 were determined for the nitric oxide product in assay solutions containing 0.1 or 100 mM KPO 4 buffer (pH 7.4), respectively, with a limit of detection of 1 μM. Acidification of these reactions (pH 2.4) generated a pink-colored product absorbing at 540 nm allowing for quantitation of total nitric oxide/nitrite levels using extinction coefficients of 38,000 and 36,900 M - 1 cm - 1 , for the assay solutions described. The limit of detection of this assay was approximately 300 nM. Using the 100 mM KPO 4 buffer system, nitrate levels were determined following reduction to nitrite using a copper-coated cadmium reagent with an extinction coefficient of 29,500 M - 1 cm - 1 and a detection limit of 0.5 μM. The utility of these assays was demonstrated in the standardization of nitric oxide-saturated cell culture media, and the release of nitric oxide by the NONOate compound DEA/NO.