We examined the expression of a recently characterized novel estrogen receptor (ER) β in 25 cases of invasive ductal carcinoma of the breast, using messenger RNA (mRNA) in situ hybridization, and compared the findings with those of ERα, to study its localization and its possible biological significance in human breast cancer. ERα and ERβ hybridization signals were both detected, predominantly in carcinoma cells and in some stromal cells, in 18 of 25 (72%) and 11 of 25 (44%) cases, respectively. The cases in which more than 25% of carcinoma cells demonstrated mRNA hybridization signals were 13 of 25 (52%) and 2 of 25 (8%) cases for ERα and ERβ, respectively. Among the cases expressing ERβ, 10 of 11 (91%) also expressed ERα mRNA; and in these 10 cases, coexpressing both ERα and β, the number of carcinoma cells expressing ERa was greater than that expressing ERβ in 9 cases. Eight cases demonstrated only ERα mRNA hybridization signals in carcinoma cells. These results indicate that ERβ is coexpressed with ERα in most ERβ-positive breast carcinoma cells, which suggests that the expression of ERβ depends on the presence of ERα in the great majority of human breast cancer. In addition, the number of carcinoma cases and/or the ratio of carcinoma cells expressing ERα was much greater than those expressing ERβ. The relative ratio of ERa and ERβ expression in carcinoma cells may be related to various estrogen-dependent biological features of human breast cancer.