The phagocytic activity and the chemiluminescence response of human polymorphonuclear neutrophils were studied with normal human serum used as opsonin for S. typhimurium and S. aureus. When serum was heated, chelated with ethyleneglycol tetraacetic acid (EGTA), or absorbed with zymosan, there were reduction of phagocytic activity for S. aureus and a comparable reduction of chemiluminescence; a 50 per cent decrease in phagocytosis by neutrophils corresponded to a 50 per cent reduction in the chemiluminescence response. In contrast, S. typhimurium was successfully phagocytized in serum treated with MgEGTA (complement--alternate pathway intact; classic pathway blocked), and chemiluminescence was reduced by only 20 per cent under the same conditions. Both neutrophil phagocytic and chemiluminescence responses for S. typhimurium were abolished when serum was heated at 56 degrees C. or adsorbed with zymosan. These findings suggest that both phagocytosis and chemiluminescence in neutrophils are interrelated, and the chemiluminescence response of neutrophils may be a reliable assay for measuring serum opsonic activity.