Human milk fortifier (HMF) is routinely added to human milk feedings to augment nutrient and caloric delivery to premature infants. We previously reported that the TGFα immunoreactive concentration in the fat, but not the aqueous compartment, of human milk is decreased with the addition of HMF[Ped Res 1997; 41(4):515A]. TGFα is detectable in HMF when reconstituted in sterile water (concentration 2 pg/mL), but not in commercially available formulas. It is unknown if HMF alters the biochemical properties, including the TGFα isoform profile, of human milk. To address this question, the TGFα isoform profiles of whole milk and its fractions were compared with the same fractions with a commercially available powered HMF (Mead-Johnson). Fifteen whole milk samples were collected from 12 mothers on postpartum days 4 through 90; gestational age of the infants ranged from 26-40 weeks. These samples were divided into 2 aliquots of 25 mL; 1 packet of HMF (0.96 g) was added to the first aliquot. Two cc of whole milk were retained for later analysis. The remainder of each sample was centrifuged at 1000 X g for 20 min at 4°C to achieve separation of the fat and aqueous fractions. Sodium taurocholate, a bile salt, was added 1:1 (v/v) to the fat for emulsion. Western blot analysis of the samples was performed with a monoclonal TGFα antibody (0.5 μg/mL; RDI) and a polyclonal secondary antibody (1:20,000; Pierce) using 15% SDS-PAGE electrophoresis- semi-dry transfer method. Isoform bands were visualized by enhanced chemilluminescence(Amersham). TGFα isoforms were detected in whole milk and its separated fractions at 6.5, 12-16, 22, 26, and 30 kD. HMF alone and HMF with sodium taurocholate had a single detectable band at 18 kD. While whole and aqueous milk samples with HMF present also showed the 18 kD band, the fat fraction samples with HMF did not have the 18 kD band. It appears that HMF differentially alters the biochemical profile of human milk with regard to TGFα concentration and the immunodetection of TGFα isoforms. What effect this alteration in human milk biochemistry has on neonatal gut function remains unknown.