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