Human milk fortifiers (HMF) are commonly added to increase caloric density and nutrient content of mother's milk given to preterm infants. While it is known that certain growth factors (IL-10, TGFα, and TGFβ) are compartmentalized within human milk, it remains unknown if HMF affects the distribution and immunoreactivity of such growth factors within those compartments. We sought to measure the concentration of immunoreactive TGFα in whole milk and its fractions following the addition of a commercially available powdered HMF (Mead-Johnson). Fifteen milk samples were collected from 12 mothers on days 4 to 90 of lactation; gestational age of their infants ranged from 26-40 weeks. Whole milk samples were divided into 2 aliquots of 25 mL; 1 packet (0.96 g) HMF was added to the first aliquot. Whole milk was centrifuged at 1000g × 20 min at 4°C to achieve separation of fat and aqueous components. Sodium taurocholate, a bile salt, was added 1:1(v/v) to the fat for emulsion. The concentration of TGFα in the whole, aqueous and fat fractions of the untreated vs. HMF-treated samples was measured by RIA (RDI; standard curve unaltered by Na taurocholate). The mean± S.D. of TGFα in unfortified whole and aqueous milk samples did not differ from HMF-treated samples: whole milk untreated 14.8±8.0 vs. HMF-treated 15.7±7.1 pg/100μL; aqueous untreated 14.0±3.5 vs. HMF-treated 14.0±2.7 pg/100μL. There was, however, a marked decrease in the concentration of immunoreactive TGFα in the fat fraction of all HMF-treated milk samples tested. The mean ± S.D. TGFα concentration in the unfortified fat samples was 98.0±69 (range 12-230) vs. 30.6±28.3 pg/100μL (range 10-122) in the fortified fat samples(mean ↓ 60%, range 5-89%). While HMF does not appear to affect the immunodetection of TGFα in the aqueous compartment of whole milk, it does appear to affect the immunodetection of TGFα in the lipid compartment. The addition of HMF to mother's milk may affect the recognition by gut epithelia of bioactive substances within the fat compartment, affecting subsequent binding and cell activation.