Neutrophils (PMN) from newborn calves generate significantly less superoxide anion (O2-) than do their adult counterparts after stimulation with direct protein kinase C agonists. To better understand this observation, we compared the activity and kinetics of NADPH oxidase in membrane fractions from phorbol 12-myristate 13-acetate-stimnlated adult and newborn PMN. After phorbol 12-myristate 13-acetate stimulation, PMN were sonicated and the membranes assayed for O2- production with increasing concentrations of NADPH. O2- production was calclated 1 and 2 min after the begianing of the reaction. At all concentrations of NADPH ased, there was no difference (p> 0.05) in O2- production between adult (n = 8) and newborn (n = 9) PMN membrane preparations. Enzyme kinetics calculations revealed no differences (p> 0.05) between age groups in Km and Vmax or in the velocity of the reactions. Determination of the protein content in the membrane pellet, however, showed that adlut PMN yielded significiantly (phigher amounts of protein (2.82 \pm 0.14 mg/ml) than did newborn PMN (1.78 \pm 0.07 mg/mL). This difference could be partly attributed to cell size; flow cytometric analysis showed that newborn PMN had a significiantly (pp< 0.01) in newborn PMN. These data collectively showed that the observed difference in O2- production between newborn and adult bovine PMN stimulated with protein kinase C agonists was not due to a difference in the activity or the kinetics of the enzyme NADPH oxidase, and that PMN from newborn calves had a significantly smaller diameter, volume, and surface area than did adnlt PMN. These size differences could play a role in the O2- generating deficit of newborn bovin PMN. (Pediatr Res28:327–331,1990)