Using a metabolic test of hyphal viability, the interaction between neutrophils and Aspergillus hyphae was investigated over a broad range of hyphae-to-neutrophil ratios. Normal neutrophils were found to damage hyphae whereas neutrophils from patients with both chronic granulomatous disease (CGD) and myeloperoxidase (MPO) deficiency did not. Further, both azide and catalase + superoxide dismutase inhibited the ability of normal neutrophils to damage hyphae, suggesting that this damage is mediated by products of the respiratory burst and by the MPO-halide system. Also, mixtures of small numbers of normal neutrophils with larger numbers of CGD neutrophils (range, 1:5 to 1:15) damaged hyphae more efficiently than either population of cells alone. Further, mixtures of CGD and MPO-deficient neutrophils, neither of which alone could efficiently damage hyphae, were able to damage the hyphae almost as well as a comparable number of normal neutrophils. These data demonstrate that intact neutrophils can cooperate to synergistically damage Aspergillus hyphae, possibly by extracellular mixing of hydrogen peroxide and MPO.