It has been reported that the biological activities of several beta-glucans vary due to differences in their physicochemical properties. In this study we investigated the ability of beta-glucans to trigger H2O2 production and activate signaling pathway on peritoneal macrophages. The most effective beta-glucan tested on H2O2 production was zymocel which was a particulate preparation from the yeast cell wall. In contrast, gel-forming beta-glucans which are known as immunoenhancers did not trigger the H2O2 production by macrophages at all. To identify the related signaling pathway for the particulate beta-glucans-triggered H2O2 production, several inhibitors were applied. Hydrogen peroxide production triggered with phorbol myristate acetate was inhibited by pretreatment of macrophages with H-7, a protein kinase C inhibitor. However, beta-glucans-triggered H2O2 production was not affected by H-7. The results suggested that genistein-sensitive tyrosine kinase and bromophenacyl bromide-sensitive phospholipase A2 participated in the particulate beta-glucans-triggered H2O2 production, although the phagocytosis of particulate beta-glucans was not inhibited by either reagents. In conclusion, gel-forming (1-->3)-beta-D-glucans-induced activation was not sufficient to trigger H2O2 on macrophages, and pathways for particulate beta-glucans-triggered H2O2 production were regulated differently from those for phagocytosis of beta-glucans.