Enteral feeding, in particular with formula feeds, is associated with necrotizing enterocolitis (NEC). In this study, we have examined, in the systemic and mucosal immune compartments, for evidence of bovine milk antigen sensitization in infants with NEC. Eleven newborns with Bell's staging 2-3 NEC [median post-conceptional age 31 wk (range 27-41 wk)], 21 neonatal controls [33 (28-40) wk] and 15 infants undergoing intestinal resection or mucosal biopsy for non-inflammatory conditions [39 (34-42) wk] were studied. Spontaneous and antigen or mitogen elicited interferon-gamma (IFN-gamma) [T-helper type I (Th1)], interleukin (IL)-4 and IL-5 [T-helper type II (Th2)] responses were enumerated using single-cell enzyme-linked immunospot (ELISPOT) assay in peripheral blood (PBMC) or lamina propria mononuclear cells. NEC infants, compared with controls, showed a significant elevation in baseline PBMC cytokine secreting cells, vigorous mitogen responses (20- to 120-fold increase) for IFN-gamma, IL-4 and IL-5 (p < 0.001), strong responses to beta-lactoglobulin (betalg) (IFN-gamma > IL-4/IL-5, p < or = 0.001), and somewhat smaller casein responses. Similarly, in the lamina propria, a small but significant increase in spontaneous cytokine-secreting cells was detected in NEC infants (p < 0.01), with an IFN-gamma/IL-4 predominant phytohemagglutinin (PHA)/concanavalin-A (ConA) response. Three of nine NEC infants (but no controls) also showed a positive ELISPOT response to betalg (IFN-gamma only) but none to casein. We have thus demonstrated significant cow's milk protein (CMP) sensitization in NEC, at least in the systemic compartment (mixed Th1/Th2), with minimal mucosal activation in some cases. These novel findings provide a potential mechanism for a direct contributory role of CMP in the pathogenesis of NEC.