Generation of toxic oxygen metabolites through the activity of the respiratory burst is crucial to the antimicrobial activity of neutrophils (PMNs). We have previously shown that cord blood PMNs generate increased amounts of superoxide anion (O2−) compared to cells from adults. To determine the basis for this increased respiratory burst activity, we measured O2− generation in subcellular fractions of PMNs. Blood was obtained from 6 placentas of term, vaginally delivered infants and 6 adults; and PMNs were separated by standard techniques with LPS-free reagents and disrupted by nitrogen cavitation. Plasma membrane-rich fraction (MRF) was separated by differential centrifugation. O2− was measured as superoxide dismutase inhibitable cytochrome c reduction at various concentrations of NADPH. Kinetic parameters were calculated by Lineweaver-Burk analysis. The apparent Km for NADPH of cord blood PMNs was increased (numbers are mean ± SEM, p<0.05) compared to adult PMNs, but not to a level implying abnormal cell function. Vmax was strikingly increased (p<0.05) in cord blood samples. The data suggests a small difference in affinity for NADPH in cord blood PMNs. The increased Vmax may be related to “priming” of the oxidase, possibly as a result of partuition; and this could explain the increased respiratory burst activity of intact cord blood PMNs.