Necrotizing enterocolitis (NEC) is a serious medical and surgical problem affecting newborn infants, particularly prematures. The pathologic processes contributing to NEC are unknown, however inflammatory mediators are believed to play a key role. Defensins are cystine rich peptides with broad spectrum antimicrobial activity produced by Paneth cells in the small intestine. Enteric defensin 5 and 6 (HD5, HD6) mRNA are constitutively expressed in adult Paneth cells at high levels, but detected at significantly lower levels during fetal development. The reduced expression of human enteric defensin mRNA in the premature neonate could be important in the pathophysiology of NEC. To study the possible role of defensins in NEC, we analyzed small intestinal samples from 6 newborn infants with NEC and 6 newborn controls with either meconium ileus or duodenal atresia. Expression of HD5 and HD6 mRNA, as well as collagen IVa mRNA was determined using in situ hybridization. Enteric defensin mRNA was increased in all cases of NEC compared to controls, while collagen IVa mRNA remained unchanged. In contrast, immunohistochemical studies on these tissue samples using antibody to HD5 showed similar levels of HD5 protein in Paneth cells of NEC versus control patients. In conclusion: 1)the increased expression of HD5 and HD6 mRNA may be a response to local production of inflammatory mediators; 2)similar intracellular levels of HD5 may indicate release of the antimicrobial peptide to bolster local intestinal immunity. The role of defensins in neonatal susceptibility to NEC needs further investigation. (Funded by NIH AI32738, RR00240, AI32234).