The NADH oxidoreductase reaction with resazurin was most rapid at pH 6.5. FMN (10 mumol/l) markedly stimulated the reaction, and the optimal concentration of resazurin was 50 mumol/l. The oxidation of NADH by NADH oxidoreductase with resaruzin as electron acceptor gave a variable yield of fluorescent product, resorufin. The yield was pH dependent and was greatest at pH 6.5. Measurements of oxygen consumption in the reaction mixture demonstrated that dissolved O2 was an alternative electron acceptor. The increased yield of resorufin at pH 6.5 was due to more rapid reduction of resazurin rather than oxygen. In contrast, at pH 9.0 oxygen was the preferred electron acceptor. The sensitivity of assays utilizing this indicator reaction can be improved by these optimized conditions.