Epidermal growth factor (EGF) is one of trophic factors for intestinal adaptation after small bowel transplantation (SBT). Recent report indicates that nitric oxide (NO) has cytoprotective effect on bacterial translocation (BT) after SBT. We hypothesized that EGF stimulates the expression of inducible nitric oxide synthase (iNOS) gene in the graft after SBT, followed by increased production of NO, resulting in the decrease of BT. Intestinal epithelial cells (IEC-6) were treated with EGF or/and interleukin-1 (IL-1) in the presence and absence of phosphatidylinositol 3-kinase (PI3K) and EGF receptor kinase inhibitors (LY294002 and tyrphostin A25). The induction of NO production and iNOS, and its signal molecules including the inhibitory protein of NF-B (IB), nuclear factor-B (NF-B) and Akt were analyzed. IL-1 stimulated the degradation of IB and the activation of NF-B, but had no effect on iNOS induction. EGF, which had no effect on the NF-B activation and iNOS induction, stimulated the up-regulation of type 1 interleukin-1 receptor (IL-1R1) through PI3K/Akt. Simultaneous addition of EGF and IL-1 stimulated synergistically the induction of iNOS, leading to the increased production of NO. Our results indicate that EGF and IL-1 stimulate two essential signals for iNOS induction in IEC-6 cells, the up-regulation of IL-1R1 through PI3K/Akt and the activation of NFB through IB kinase, respectively. Simultaneous addition of EGF and IL-1 can enhance the production of NO, which may contribute to the cytoprotective effect of EGF against intestinal injury.