Lactadherin (formally BA46) is a major glycoprotein of the human milk fat globule (HMFG) and is also present in skim milk (1-3% of total milk protein). It has been implicated in the anti-rotavirus activity of human milk. cDNA cloning reveals an RGD cell adhesion sequence inserted into a EGF-like motif and a C-terminal domain with homology to the C1/C2 domain of human coagulation factors V and VIII. Lactadherin promotes cell attachment of breast carcinoma, monkey kidney and mouse fibroblast cells at nM concentrations and this attachment is inhibited specifically with RGD-containing peptides. Though lactadherin cDNA sequence suggests a soluble protein, it is membrane-associated, being detected on the surface of HMFGs by immunofluorescence, partitioning into the detergent phase in Triton X-114 fractionation, and associating avidly with liposomes. Its lipophilic nature is probably the result of covalently bound phospholipids or fatty acids. If the above cells, and also colon carcinoma cells and normal human lymphocytes, are incubated with skim milk or purified lactadherin in a liposome complex, lactadherin binds with a high affinity to focal adhesion-like sites, as detected with specific anti-lactadherin MoAbs. Although its specific cell ligand has not yet been identified, lactadherin probably binds to an integrin via its RGD sequence presented on a loop structure supported between two anti-parallel β-strands of the EGF-like motif. The specific binding of lactadherin to integrins on a variety of cell types, including cells of the GI tract, could be important for its anti-viral activity or could contribute to other beneficial attributes of human milk in breast-fed infants, such as promoting neonatal development of the intestinal mucosa. Alternatively, its binding to milk lymphocytes or other cells in milk could alter their immunological properties or viability, by acting as either an adhesion or anti-adhesion molecule. Supported in part by NIH Grant Nos. HD30444, CA61258, and CA39932.