Hydrogen peroxide (H2O2) inhibited phagocytosis and superoxide anion production by rat alveolar macrophages. The inhibition was irreversible and concentration and exposure time dependent. The potential relationship between H2O2-induced biochemical perturbations and impaired alveolar macrophage phagocytic functions was investigated. Alveolar macrophage viability and Fc receptor binding capacity were not affected by H2O2. There was probably no correlation between a H2O2-induced rise in cytosolic [Ca2+] ([Ca2+]i) and the impairment of phagocytosis by alveolar macrophages, as was suggested by the following findings. First, the H2O2-induced rise in [Ca2+]i could be inhibited by chelation of extracellular Ca2+, whereas the H2O2-induced impairment of phagocytosis could not. Second, the H2O2-induced rise in [Ca2+]i was reversible, whereas the impairment of phagocytosis was not. And finally, a rise in [Ca2+]i by incubation of alveolar macrophages with the calcium ionophore A23187 did not affect phagocytosis. Various experiments suggested that ATP depletion may play an important role in the H2O2 toxicity for alveolar macrophages. Comparable concentrations of H2O2 caused an irreversible decrease both in cellular ATP and in phagocytosis and superoxide production by alveolar macrophages. In addition, time course of ATP depletion and induction of impaired alveolar macrophage function were similar. In view of the fact that the strong oxidant H2O2 may react with a large variety of biological substances, possible other toxic lesions may not be excluded as underlying mechanism for H2O2-induced inhibition of phagocytic functions of alveolar macrophages.