An estrogen-induced, intensely staining peroxidase 3,3-diaminobenzidine-positive reaction product is found to be characteristic of hormone-dependent, 7,12-dimenthylbenz(a)anthracene-induced mammary tumors of the rat. This product is demonstrated in thick sections of such tumors from intact or estrogen-treated castrate rats but is not seen in tumors that are in regression due to castration or estrogen deprivation. It is, furthermore, absent from tumors whose growth is unaffected by castration. The subcellular localization of this enzyme activity is restricted mainly to the nuclear envelope and cisternae of the granular endoplasmic reticulum in addition to secretory granules. This provides the first evidence for a criterion that would allow differentiation of hormone-dependent and hormone-independent mammary cancer on histological sections and, as such, may have considerable potential as an aid in the classification of human breast cancer.