To understand the significance of estrogen receptor β (ERβ) in mammary carcinogenesis, we evaluated the expression of ERβ in preinvasive mammary tumors. The percentage of ERβ-positive epithelial or tumoral cells was assayed by quantitative immunohistochemistry using an image analyzer in 130 lesions of varying histological risk from 118 patients [71 with benign breast disease (BBD) and 59 with carcinoma in situ (CIS)] and compared with 118 adjacent histologically normal glands. Five groups of lesions with an increasing risk of invasive cancer, from BBD without hyperplasia to high-grade CIS, were studied. Results were compared with ERα and Ki67 immunostaining. The percentage of ERβ-positive cells was high (median, 85%) in “normal” mammary glands and in nonproliferative BBD and decreased significantly (P < 0.0001) in proliferative BBD without atypia and in CIS, contrasting with an inverse progression for the ERα level. In normal mammary glands, the ERβ level did not vary according to the nature of the lesion at the periphery and was significantly higher (P < 0.007) than in adjacent preinvasive lesions, except in nonproliferative BBD. The ERβ level decreased in proliferative BBD, anticipating the ERα increase, which was significant in BBD with atypia. In high-grade ductal carcinoma in situ, both ER levels were low. The ratio between ERβ and ERα was high in normal glands, and decreased significantly in proliferative lesions. ERβ staining was inversely correlated with Ki67 (r = −0.333; P < 0.001), more particularly in high-grade ductal carcinoma in situ (r = −0.57; P < 0.02). The marked and early decreased level of ERβ protein associated with other criteria of cell proliferation suggests a protective effect of ERβ against the mitogenic activity of estrogens in mammary premalignant lesions. Knowledge of the ERβ and ERα content in each preinvasive lesion should help to rationalize antiestrogen preventive therapy adapted to each individual patient.