Perinatal transmission of human immuno-deficiency virus (HIV) from infected mothers to their children occurs at rates reported as 20–50%. The role of breast feeding in perinatal transmission of viral infections has not been well established. We studied 34 milk and colostral samples obtained from HIV-seropositive and HIV-sero-negative women to determine if they contained anti-HIV activity. We found that all the samples contained a factor that inhibited the binding of HIV epitope-specific MAb to recombinant CD4 receptor molecules. The titers of inhibitory activity ranged from 1:200 to 1:10 000 and did not differ between HIV-seropositive and HIV-seronegative mothers. This milk factor also inhibited the binding of gp120 to CD4. Neither human sera nor bovine milk exhibited appreciable inhibitory activity. Fractionation of human milk indicated that the inhibitory activity was confined to the macromolecular fraction; little activity was found in isolated milk lipids or oligosaccharides. Chromatographic procedures indicated that the active macromolecule has an isoelectric point of 9.3–9.6. The active material did not bind to concanavalin A; however, the activity was partially destroyed by chemical and enzymatic treatments that removed sulfated residues. The active material may thus be a sulfated protein, glycoprotein, mucin, or glycosaminoglycan that inhibits the binding of CD4 to HIV envelope glycoproteins. The role of this factor in the natural history of HIV infection in infants and children should be the subject of additional investigations. (Pediatr Res31: 22–28, 1992)