In this study we have investigated the effects of serum (10, 20, and 40% final concentrations) on the activity of NADPH-oxidase and energy metabolism of activated human neutrophils in vitro. Neutrophils were stimulated with FMLP, PMA, or opsonized zymosan in the presence and absence of serum, and generation of reactive oxidants by intact cells was measured using lucigenin-enhanced chemiluminescence (LECL). This method was also used to measure NADPH-oxidase activity in purified membrane preparations from neutrophils activated with PMA in the presence or absence of serum. Cellular ATP levels and activities of the various glycolytic enzymes were assayed using CL and spectrophotometric procedures, respectively. Inclusion of serum with neutrophils during exposure to the various stimuli of membrane-associated oxidative metabolism caused significant enhancement of the LECL responses of intact cells as well as of the activity of NADPH-oxidase in purified membranes prepared from PMA-activated neutrophils. In the absence of serum, the ATP levels and activity of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), but not the other glycolytic enzymes, were decreased in activated neutrophils, while inclusion of serum preserved neutrophil ATP levels and activity of G3PDH. Serum supplementation of the cell-suspending medium appears to promote optimum activity of NADPH-oxidase in stimulated neutrophils by preventing premature, oxidative inactivation of cellular energy metabolism.