Human milk contains many cytokines, including transforming growth factor-beta isoforms (TGFβ1 and β2). TGFβ appears to control growth and differentiation of neonatal intestinal epithelia, and to act in the repair of the gut following injury. It is thought that the bioavailability of these factors can be affected by their compartmentalization in milk. While an increase of certain bioactive factors within the fat compartment of milk has been shown, it is unknown if TGFβ1 shares similar distribution properties. We sought to determine if there is a differential concentration of TGFβ1 in the human milk fat layer compared with whole milk and its aqueous fraction. Thirty-six whole milk samples and an additional 13 milk fat samples were collected from 35 lactating mothers on postpartum day 3 to 6 mo.; gestational age of their infants at delivery ranged from 32 to 42 weeks. Whole milk was centrifuged at 1000g× 20 min at 4°C to achieve separation and isolation of its fat, aqueous and cellular components. Tris buffered saline (TBS; 200 μL) with 10μL Tween-20 was added to the fat for emulsion. The concentration of acid-activated TGFβ1 in whole milk, and its aqueous and fat components was measured by ELISA (Promega; sensitivity 25 pg/ml sample; cross-reactivity with other TGFβ species of <5%). TGFβ1 was detected in 19/36 whole milk samples (95 ± 80 pg/mL; range 27-345); 4/26 aqueous samples (64 ± 32 pg/mL; range 30-93); and 31/48 fat samples (281 ± 226 pg/mL; range 34-958). The concentration of TGFβ1 expressed per gram of fat in 8 of 13 samples was 1.5± 1.4 pg/gm fat (range 0.3 - 4.3). When present in whole milk, TGFβ1 was found at higher concentrations in the fat layer with lower/nondetectable levels in the aqueous compartment of all samples tested. This has implications regarding the differential distribution and bioavailability of TGFβ1 in the newborn gut.