The time-dependence of the reaction of human erythrocyte diaphorase activity has been studied by the use of NADH2 and ferricyanide as substrates. Reaction was found to be first-order with respect to NADH2 concentration, and zero-order with respect to ferricyanide concentration. These findings indicate that human erythrocyte diaphorase has a Km value for NADH2 by far higher than, and for ferricyanide by far lower than, the concentration of the substrates used, i.e. 0.1 and 0.2 mmol/l, respectively. The diaphorase activity determination method, described in the present communication, has been used in 19 healthy adults and children. Diaphorase activity was found to be 7.29 +/- 3.69 1 SD mumol NADH2 oxidized/ml packed cells per min, at 25 degrees C, and pH 7.00.