Necrotizing enterocolitis (NEC) seems to result from the inflammatory response of an immature intestine. Human milk is protective against NEC via an unknown mechanism. We hypothesized that specific factors found in human milk would decrease stimulated IL-8 secretion in intestinal epithelial cells. HT29-cl19A and Caco2 cells were compared with the fetal human primary intestinal epithelial cell line H4 and temperature-sensitive conditionally immortalized fetal human intestinal (tsFHI) cells. Cells were pretreated with transforming growth factor-beta (TGF-beta), erythropoietin (Epo), IL-10, or epidermal growth factor (EGF) at physiologic concentrations before stimulation with tumor necrosis factor-alpha (TNF-alpha) or IL-1beta, and then IL-8 was measured by ELISA. The fetal cells produced significantly more IL-8 when stimulated by TNF-alpha or IL-1beta. There were also differences in the pattern of alteration of IL-8 secretion by human milk factors. In HT29-cl19A cells, IL-10 inhibited TNF-alpha-stimulated IL-8 secretion by 52%, and EGF increased secretion by 144%. In H4 cells, TGF-beta1 and Epo inhibited TNF-alpha-stimulated IL-8 secretion to control levels, and EGF increased secretion by 29%. IL-1beta-stimulated IL-8 secretion was inhibited 25% by TGF-beta1 in Caco2 cells and in H4 cells was inhibited by TGF-beta1, Epo, and TGF-beta2. TsFHI cells confirmed H4 cell results. Fetal human enterocytes have an exaggerated IL-8 secretion in response to TNF-alpha and IL-1beta. TGF-beta and Epo decrease this stimulated IL-8 secretion, which may partially explain the protective effect of human milk in NEC.