Membrane-bound antigens were solubilized and purified from the continuously growing human mammary adenocarinoma cell line SW527. Two distinct breast tumor-associated antigens (BTA-1 and BTA-2) were obtained, as well as purified HLA antigens. The molecular weights of BTA-1 and BTA-2 were estimated at 112,000 and 65,000, respectively, by analytical polyacrylamide gel electrophoresis. The two mitogens also differed in carbohydrate content and hexose/pentose ratio. Antibodies to these antigens, raised in rabbits, were shown to be directed against SW527 surface markers by an immune adherence hemagglutination assay. Purified BTA-1 and BTA-2 reacted with rabbit antibodies to SW527 and with antibodies to a tumor-associated antigen previously isolated from tumor tissue freshly obtained from a patient with mammary adenocarcinoma, but did not react with anti- β 2-microglobulin. By indirect immunofluorescence assay, SW527 surface markers were shown to be recognized by antibodies in the serum of a patient with mammary adenocarcinoma; this reactivity was blocked by treatment of the serum with the tumor-associated antigen purified from fresh tissue. These results indicate that the antigens isolated from tumor cells in tissue culture and directly from human tumor tissue have common immunological properties.