Investigators disagree on the amount of hydrogen peroxide (H2O2) released by resting and stimulated alveolar macrophages. The method commonly used to measure H2O2 release involves horseradish peroxidase (HRP)-catalyzed oxidation of scopoletin by H2O2. We describe an artifact in this method that may explain the seemingly inconsistent data reported by other investigators. Release of H2O2 and luminol-catalyzed chemiluminescence are stimulated in rat alveolar macrophages by type II HRP at concentrations normally used in the HRP-scopoletin method. The amount of H2O2 released depends upon the length of time the cells are preincubated at 37.5 degrees C and the time at which type II HRP is added. After stimulation with type II HRP, the cells do not release additional H2O2 upon exposure to zymosan particles. Myeloperoxidase, an alternative catalyst to type II HRP, does not stimulate H2O2 release and, therefore, can be used to measure H2O2 release from rat alveolar macrophages. Using myeloperoxidase, resting H2O2 release is negligible; after zymosan stimulation, 6.14 (+/- 0.87) X 10(-6) nmoles/cell X 10 min is released. In addition, more pure HRP preparations (types VI, VII, VIII, and IX) do not stimulate alveolar macrophages to release H2O2 and can be used to monitor zymosan-induced H2O2 release. As our data indicate that type II HRP stimulates H2O2 release from rat and guinea pig alveolar macrophages, it is not the catalyst of choice for this assay. In conclusion, our data explain the conflicting results found in the literature and indicate that rat alveolar macrophages release minimal amounts of H2O2 at rest and can be stimulated by zymosan.