Interleukin (IL)-1β, a multifunctional cytokine, is associated with inflammatory gastric mucosa, but the responses of gastric epithelial cells stimulated by IL-1β are not known. We determined whether IL-1β activates the two subfamilies of mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinases (ERKs) and c-Jun NH2-terminal kinases (JNKs), in rat gastric epithelial cells (RGM1) using in-gel kinase assays. In addition, we examined the mechanism(s) underlying their signaling pathways and the effect on proliferation of these cells. IL-1β (0–5 × 103 pg/ml) dose dependently induced activation of ERKs (p44ERK and p42ERK) and JNKs (p46JNK and p55JNK) in RGM1 cells; maximal activities were attained with 1,000 pg/ml of IL-1β. These activities were increased with time, and were maximal 20 min after stimulation with IL-1β (100 pg/ml). Pretreatment with neutralizing antibody against IL-1β inhibited IL-1β-induced activation of ERKs and JNKs. Genistein (100 μM), a tyrosine kinase inhibitor, and GF109203X (2 μM), a protein kinase C inhibitor, inhibited the IL-1β-induced activation of ERKs and JNKs. Six- hour pre-incubation with IL-1β inhibited proliferation of these cells by 40% at 24 h of incubation, but the inhibition was recovered at 48 h. These findings suggest that IL-1β activated ERKs and JNKs in rat gastric epithelial cells and inhibited cell proliferation, possibly via the specific receptor for IL-1β. Activation of MAP kinases by IL-1β may be mediated by tyrosine kinase and protein kinase C.