Using an assay that measured superoxide dismutase-inhibitable nitro blue tetrazolium reduction, we studied superoxide (O2-) production by a cell-free system from human granulocytes. At 40 muM NADPH and a protein concentration of 0.12 mg/ml, lysates prepared from human granulocytes formed O2- at a rate of 18. 4 +/- 4.6 SE nmol/ml reaction mixture per donor, but not with glucose-6-phosphate, 6-phosphogluconate, glyceraldehyde-3-phosphate, sodium lactate, glutathione, or ascorbic acid. The Km's for NADPH and NADH were 8.6 +/- 4.6 muM and 0.83 +/- 0.30 mM, respectively, suggesting that NADPH is the physiological electron donor in this system. O2- production was not inhibited by 1mM KCN. The rate of O2- production by the cell-free system was comparable to the rate of O2-production by an equivalent quantity of intact granulocytes incubated under similar conditions. O2- production by lysates from granulocytes preincubated with serum under conditions previously shown to stimulate O2- production in the intact cells was no different than its production by lysates from unstimulated cells. O2- production at 0.2 mM and 0.02 mM NADPH by lysates from the granulocytes of two patients with chronic granulomatous disease was similar to O2- production by control lysates. This finding was interpreted in terms of the possibility that the metabolic lesion in chronic granulomatous disease may lie outside the oxygen-metabolizing enzyme system of the granulocyte, or alternatively, that the granulocytes may contain two O2- - forming enzymes, one of which is inactive in chronic granulomatous disease.