The enzyme responsible for the respiratory burst in human neutrophils is a membrane-bound oxidase that catalyzes the reduction of oxygen to O2- at the expense of a reduced pyridine nucleotide. We describe further properties of the solubilized oxidase. The rates of O2- production and NADPH consumption are consistent with the stoichiometry: 2 O2 + NADPH leads to 2 O2- + NADP The enzyme is highly specific for oxygen, failing to reduce several artificial electron acceptors including ferricyanide. FAD, an essential cofactor, binds tightly to the enzyme, as indicated by a Km of 61 nM. A requirement for a free --SH group is suggested by a 2-fold increase in activity in the presence of low concentrations of dithiothreitol; the higher dithiothreitol concentrations lead to inhibition. The solubilized enzyme is highly unstable, losing one-half its activity after 1 h at room temperature. Loss of activity is accelerated 2- to 3-fold by salts and EDTA. The substrate analog 2',5'-ADP is similar to other salts in its effect on the inactivation rate. ATP, on the other hand, causes loss of activity in seconds, raising the possibility that ATP is a physiological regulator of the catalytic activity of the enzyme.