In uremia, diminished reactive oxygen intermediate production is an important consequence of impaired neutrophil function. The effects of guanidino compounds, which are known uremic toxins, on neutrophil reactive oxygen intermediate production in vitro were studied. Neutrophils from healthy volunteers were exposed for 3 h to individual guanidino compounds or mixed guanidino compounds (GCmix), at concentrations observed in uremic plasma. After removal of the guanidino compounds, the neutrophils were activated by adhesion, N-formylmethionylleucylphenylalanine, phorbol myristate acetate, or opsonized zymosan, and superoxide production was measured by monitoring lucigenin chemiluminescence. The direct effects of guanidino compounds on superoxide production in activated neutrophils were also measured. The energy status (ATP and creatine phosphate), antioxidant status (total glutathione), and glycolytic flux (lactate production) were measured. GCmix pretreatment decreased superoxide production in activated neutrophils (activated by N-formylmethionylleucylphenylalanine or zymosan) by 50% (P < 0.01), decreased ATP concentrations by 60% (P < 0.05), and inhibited glycolytic flux (lactate production) by 45% (P < 0.01) but did not alter glutathione concentrations. Simultaneous GCmix exposure and activation did not inhibit NADPH oxidase activity in cell lysates but inhibited superoxide formation in zymosan-activated intact neutrophils; this inhibition was reversed after removal of the guanidino compounds. Guanidinosuccinic acid, guanidinopropionic acid, and guanidinobutyric acid, when tested individually, were each as potent as GCmix. The inhibition of neutrophil superoxide generation by guanidino compounds results from decreased energy status. Micromolar concentrations of guanidino compounds significantly inhibit neutrophil metabolism, with serious implications for the functions of neutrophils in host defenses.