The mechanisms by which hydrogen peroxide and, for comparison, 4-beta-phorbol-12-myristate-13-acetate (PMA) stimulate release of radiolabeled arachidonic acid (14C-AA) in cultured intestinal epithelial cells (INT 407) were investigated. Both hydrogen peroxide and PMA caused a rapid (3 min) and dose-related intracellular release of free 14C-AA, followed by a dose- and time-dependent release of 14C-AA into the extracellular medium, but hydrogen peroxide was about 50,000 times less effective than PMA in releasing 14C-AA. No 14C-AA was released on stimulation with 4-alpha-phorbol-12,13-di-decanoate (PDD), a phorbol ester that does not activate protein kinase C. The 14C-AA release was reduced by the phospholipase A2 inhibitors nordihydroguaiaretic acid and 4-bromophenacyl bromide and by the calmodulin/protein kinase C inhibitor trifluoperazine and the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). However, H-7 was less effective than the other inhibitors in reducing the hydrogen peroxide-stimulated 14C-AA release. The hydrogen peroxide-stimulated, but not the PMA-stimulated, rapid (3 min) 14C-AA release was associated with an increased influx of extracellular calcium. Stimulation of the cells with PMA resulted in phosphorylation of a cellular protein of about 32 kDa, whereas no phosphorylation of this protein was detected after stimulation with hydrogen peroxide. Taken together, these findings indicate that (i) both PMA and hydrogen peroxide may stimulate phospholipase A2-mediated AA release from human intestinal epithelial cells; (ii) this stimulation is brought about via protein kinase C and calmodulin-mediated events; (iii) PMA-stimulated 14C-AA release is associated with phosphorylation of a 32-kDa protein, possibly lipocortin, whereas the hydrogen peroxide-stimulated release is not; and (iv) calmodulin is more important for the hydrogen peroxide-stimulated 14C-AA release than is protein kinase C. The possibility that hydrogen peroxide-evoked AA release may contribute to the mucosal abnormality in Crohn's disease is discussed.