Using a multiplex reverse transcription-PCR assay, we compared the relative expression of estrogen receptor (ER) alpha and ER-beta mRNA between adjacent samples of normal breast tissue and matched primary breast tumors obtained from 18 different patients. Within this cohort, 7 tumors were ER negative, and 11 tumors were ER positive, as determined by the ligand binding assay. No differences in the ratio of ER-alpha:ER-beta expression were observed in the ER-negative cohort. However, in the ER-positive cohort, a significantly (P < 0.02) higher ER-alpha:ER-beta ratio was observed in the tumor compared with that of the normal tissue component. Our data revealed that the increase in the ER-alpha:ER-beta ratio was due primarily to a significant (P < 0.05) increase in ER-alpha mRNA expression in conjunction with a lower ER-beta mRNA expression in the tumor compared with that of the normal compartment in some, but not all, ER-positive cases. These results suggest that the role of ER-alpha- and ER-beta-driven pathways and/or their interaction change during breast tumorigenesis.