The Bowman-Birk protease inhibitor (BBI) is a soybean-derived protease inhibitor with anticarcinogenic and anti-inflammatory properties. BBI has previously been shown to suppress the release of superoxide anion radicals from purified polymorphonuclear leukocytes. In the present study we evaluated the effect of BBI on the production of superoxide anion radicals in differentiated HL-60 cells. HL-60 cells are human lymphocytic cells that acquire neutrophil-like characteristics when treated with dimethyl sulfoxide or tetradecanoyl phorbol acetate. Superoxide anion radical production by differentiated HL-60 cells was measured in the presence of various concentrations of BBI or BBI concentrate, a soybean extract containing high levels of BBI. BBI was observed to suppress superoxide anion radical production by differentiated HL-60 cells in a dose-dependent manner. Extracts of differentiated HL-60 cells were also observed to produce superoxide anion radicals, but this activity was not affected by the presence of BBI. These results suggest that BBI inhibits superoxide anion radical generation in HL-60 cells but does not act as a simple free radical scavenger.