A procedure for quantitative analysis of nitric oxide production by cultured mammalian cells based on nitrite ion quantitation has been optimized. The Griess assay adapted for microvolumes was found to yield optimal results when the NO3- ions formed from NO2- ions in the reaction medium were reduced by nonactivated cadmium prior to the assay to form the analyte NO2-. The assay was verified for the culture of C6 rat glioma cells. The assay developed is suitable for quantitative determination of nitrogen oxide in 96- and 48-well cell culture plates; the detection limit for NO2- was 2.1 ± 0.1 μM and that for NO3- was 2.9 ± 0.1 μM.