The present study was undertaken to detect the spontaneous mammary tumor-associated antigen ( MTAA ), and to find the cross-reacting antigen in chemically-induced mammary tumor. The antisera against spontaneous mammary tumor were raised in the WAF1 rats of the same strain and tested for the detection of tumor-associated soluble antigen of mammary tumor induced by N-ethylnitrosourea (ENU) and N-butylnitrosourea ( BNU ). The MTAA was found in the extract of spontaneous mammary tumor by the double immunodiffusion test, while it was not found in the extract of normal and fetal tissues, hyperplastic mammary gland, spontaneous fibroadenoma, and chemically-induced mammary tumor. On the other hand, the MTAA was not detected in the other types of tumors induced by ENU or BNU , i.e. gastric cancer, intestinal tumor, brain tumor, kidney tumor, bladder tumor, hemangioma, rhabdomyosarcoma, or leukemia. The spontaneous MTAA could not be detected in the spontaneous mammary tumor of C3H mice or human breast cancer either. The MTAA was extracted effectively by 3 M KC1. Furthermore, the MTAA was found in the cytoplasm of continuous established mammary tumor cell line ( SpMT -1) by the immunofluorescence test.