Free oxygen radicals are involved in the pathogenesis of necrotizing enterocolitis (NEC) in premature infants. The stress-activated p38 mitogen-activated protein kinase (MAPK) has been implicated in gut injury. Here, we found that phosphorylated p38 was detected primarily in the villus tips of normal intestine, whereas it was expressed in the entire mucosa in NEC. H2O2 treatment resulted in a rapid phosphorylation of p38 MAPK and subsequent apoptosis of rat intestinal epithelial (RIE)-1 cells; this induction was attenuated by treatment with SB203580, a selective p38 MAPK inhibitor, or transfection with p38 alpha siRNA. Moreover, SB203580 also blocked H2O2-induced PKC activation. In contrast, the PKC inhibitor (GF109203x) did not affect p38 activation, indicating that p38 MAPK activation occurs upstream of PKC activation in H2O2-induced apoptosis. H2O2 treatment also decreased mitochondrial membrane potential; pretreatment with SB203580 attenuated this response. Our study demonstrates that the p38 MAPK/PKC pathway plays an important role as a pro-apoptotic cellular signaling during oxidative stress-induced intestinal epithelial cell injury. (c) 2006 Elsevier Inc. All rights reserved.