Neuronal and inducible NO synthase (nNOS and iNOS) play a protective and damaging role, respectively, on the intestinal neuromuscular function after ischemia and reperfusion (I/R) injury. To uncover the molecular pathways underlying this dichotomy we investigated their possible correlation with orthodenticle homeobox proteins OTX1 and OTX2 in the rat small intestine myenteric plexus after in vivo I/R. Homeobox genes are fundamental for the regulation of the gut wall homeostasis both during development and in pathological conditions (inflammation, cancer). I/R injury was induced by temporary clamping the superior mesenteric artery under anaesthesia, followed by 24 and 48 hours of reperfusion. At 48hr I/R intestinal transit decreased and was further reduced by NPLA, nNOS selective inhibitor. By contrast this parameter was restored to control values by 1400W, iNOS selective inhibitor. In longitudinal muscle myenteric plexus (LMMP) preparations, iNOS, OTX1 and OTX2 mRNA and protein levels increased at 24hr and 48hr I/R. At both time periods, the number of iNOS and OTX immunopositive myenteric neurons increased. nNOS mRNA, protein levels and neurons were unchanged. In LMMPs, OTX1 and OTX2 mRNA and protein up-regulation was reduced by 1400W and NPLA, respectively. In myenteric ganglia OTX1 and OTX2 staining was superimposed with that of iNOS and nNOS, respectively. Thus in myenteric ganglia iNOS and nNOS-derived NO may promote OTX1 and OTX2 up-regulation, respectively. We hypothesize that the neurodamaging and neuroprotective roles of iNOS and nNOS during I/R injury in the gut may involve corresponding activation of molecular pathways downstream of OTX1 and OTX2.