Studies were performed to characterize the previously reported particulate O2--forming system from human neutrophils. Of eight reducing agents examined, including glutathione, ascorbic acid, and intermediates of the glycolytic and hexose monophosphate shunt pathways, only the pyridine nucleotides could serve as electron donors. At 0.1 mM pyridine nucleotide, O2- production was relatively independent of pH. The Km for NADH was approximately 0.7 mM regardless of pH, while with NADPH the Km varied from 0.02 mM at pH 6.0 to 0.3 mM at pH 7.5. The molar ratio of NADPH oxidized to O2- produced was consistent with the reaction: NADPH + 2 O2- leads to NADP+ H+; the product nucleotide was shown enzymatically to be NADP. O2- production was not inhibited by CN-, Na-, EDTA, or 1,10-phenanthroline. Particulate O2- production accounted for 35% of the oxygen taken up during the respiratory burst by an equivalent number of intact neutrophils. Greatly diminished O2- production was seen with particles prepared from cells obtained from three patients with chronic granulomatous disease, with 2.5 mM NADPH as electron donor. With 5.0 mM NADH similar observations were made with particles from two of the patients, but with this nucelotide, O2- production was only slightly reduced in the third case. The evidence available suggests that this particulate O2- -forming system is the one responsible for the respiratory burst in activated neutrophils. The relationship between this system and other O2- -forming system found in human neutrophils is discussed.