Colostrum and milk contained high concentrations of thrombospondin, although the concentration relative to total protein content decreased as lactation was established. Thrombospondin occurred in the aqueous phase of milk rather than as a component of the milk fat globule membrane. It could be purified from colostrum using established procedures after removal of lipid from the starting material. The intact protein had a molecular weight of 450 kd, but the product contained small peptides, perhaps as a result of proteolytic activity in the colostrum. Thrombospondin from goat colostrum displayed a different proteolytic fragmentation pattern from thrombospondins isolated from three human sources, but this could be a species- rather than tissue-specific difference. Breast cancer cytosols contained significantly more thrombospondin than cytosols from normal tissue or benign dysplasias. Thrombospondin levels in a variety of breast secretions all fell within the range found in colostrum and milk, as did the fluids from Na+ (group II) breast cysts. K+ (group I) cysts, however, contained fluids with low thrombospondin concentrations, eliminating apocrine cells as the source of thrombospondin in the breast.