We previously demonstrated that certain biologic activities in human milk were partially blocked by antibodies directed against human tumor necrosis factor-α (TNF-α). In this study, immunochemical methods were used to verify the presence of TNF-α in human milk obtained during the first few days of lactation. Gel filtration revealed the presence of TNF-α by RIA in molecular weight fractions between 80 and 195 kD. TNF-α could not be detected consistently by conventional Western blotting or cytotoxic assays. Although immunoreactive bands were detected by a Western blot-125I protein A technique in TNF-α-positive fractions from gel filtration, those bands proved to be nonspecific. TNF-α in milk was reliably quantified by the competitive RIA. Those studies revealed that the concentrations of TNF-α in milk were 620 ± 183 pg/mL. Although RNA to TNF-α was detected in milk leukocytes by Northern blotting, little TNF-α was found in those cells before or after stimulation with N-formyl-l-methionyl-l-leucyl-l-phenylalanine or 4β-phorbol-12β-myristate-13α-acetate. The origin of this cytokine in human milk remains unclear. Nevertheless, this study suggests that TNF-α is present in early human milk in sufficient quantities to exert possible biologic effects upon the mammary gland of the mother or the immune system of the infant. (Pediatr Res31: 79–33, 1992)