The newborn GI tract undergoes extensive maturational changes in the weeks following birth. Activation of the epidermal growth factor receptor (EGFr) following its binding of EGF or TGFα is thought to play a major role in that process. Both growth factors are present in human milk. While milk-derived EGF has been shown to induce gut epithelial proliferation in bothin vitro and in vivo models, the role of TGFα is less clear. We devised an in vitro model to compare the effect of recombinant, human milk- and HMM-derived TGFα on small intestinal cell proliferation. A fetal small intestinal cell line with epithelial characteristics (FhS-74; American Tissue Type Co.) was grown to confluence in the presence of Hybri-Care medium with EGF (30 ng/ml medium). The cells were removed from flasks following Trypsan-EDTA exposure, washed in EGF-free medium, then placed in culture in EGF-free medium at a concentration of 104 cells/well (60% confluence; 96-well plates). After 24 hr, either recombinant TGFα (0.01-50 ng/mL media), human milk supernatant (0.5 -1 ng TGFα/mL) or HMM media (20-100 pg TGFα/mL) was added to separate wells (5 wells/condition) in the presence or absence of 1° monoclonal TGFα antibody (RDI; 1 μg/mL media) for 24 hr. Cell proliferation was then measured (CellTiter 96-AQ, Promega) and expressed as% above control. A positive dose-response effect on cell proliferation by all three agents that paralleled TGFα concentration was observed. This effect was decreased in the presence of 1° TGFα antibody: 22 ± 7.1% decline of TGFα-stimulated proliferation; 25 ± 3.1% decline of HM-stimulated growth; and 27.6 ± 3.2% decline of HMM media-stimulated growth at all concentrations tested. With removal of the 1° antibody, cell proliferation returned to control levels. Binding of the TGFα present in human milk and HMM media led to a consistent decrease in in vitro gut epithelial proliferation without affecting cell viability. The proliferative effect of human milk on the gut appears to be due, in part, to TGFα.