Human milk contains growth factors and cytokines which may be important for neonatal intestinal development, including EGF, TGFα, and IGF I and II. This is the first description of vascular endothelial growth factor (VEGF) in human milk. Freshly pumped milk samples at various postpartum ages were collected. Whole milk was centrifuged at 1000g × 20min at 4°C to separate the aqueous, fat and cellular components. Human milk leukocytes, the majority of which were macrophages, were then incubated in serum-free media for 24 hrs in the presence or absence of Concanavalin A (10μg/mL media); media was collected, then analyzed by Western blot for VEGF. Small intestinal cell (FHs-74) proliferation studies were performed using CellTiter®-96 AQ(Promega) assay. The concentration of VEGF in the milk samples was measured by Quantikine ELISA (R & D Systems). Term human milk samples in the first three days of life contained 76±15 ng/mL of VEGF, which is approximately 300 times the concentration in adult serum and 100 times the concentration in term cord blood serum. VEGF was demonstrated primarily in the aqueous phase of milk by both ELISA and Western blot analysis, with very little VEGF present in the fat component of milk. In reduced breast milk samples, VEGF was present as 14.5, 16.5, 23, and 28 kD subunits, and as 14.5, 16.5, 18 and 23 kD subunits after N-deglycosylation. Activated human milk macrophages secreted VEGF into conditioned media as determined by Western blot. VEGF (100 ng/mL) did not stimulate cell division of FHs-74 cells. VEGF is known to be a potent and specific mitogen for vascular endothelial cells as well as a vascular permeability factor. Its role in human milk and maturation of the neonatal gut has yet to be determined.