Tumor necrosis factor-alpha (TNF) has been shown to induce intestinal necrosis in animals. Moreover, plasma TNF levels are elevated in patients with necrotizing enterocolitis. Thus, it is possible that TNF plays a role in the pathogenesis of NEC. In the present study we used in situ hybridization (with human TNF riboprobes) to localize TNF transcripts in the intestinal tissues from normal biopsies and NEC patients. We found that in normal intestine a small amount of TNF mRNA was present only in Paneth cells. In contrast, in the acute stage of NEC, a high amount of TNF transcripts was detected in Paneth cells as well as in infiltrating eosinophils. In one case that showed infiltrating macrophages, TNF mRNA was also detected in these cells. Resident macrophages in the lamina propria and other inflammatory cells were negative for TNF transcripts. Our results suggest that: 1) Paneth cells are the major source of TNF transcripts in normal intestine, and 2) there is a marked increase in TNF mRNA formation in Paneth cells, as well as in infiltrating eosinophils and macrophages in patients with NEC. TNF-containing cells may play an important role in the pathophysiology of NEC.