The recent cloning of a second estrogen receptor (ER), designated ERbeta, has prompted a reevaluation of the role of ERs in breast cancer. We have developed and validated a real-time RT-PCR assay to quantify ERalpha and ERbeta gene expression at the mRNA level in a series of 131 patients with unilateral invasive primary breast cancer. Although ERbeta expression showed wide variations in tumor tissues, its range (nearly three orders of magnitude) was smaller than that of ERalpha (nearly four orders of magnitude), suggesting that ERbeta is more tightly controlled than ERalpha. We observed a negative correlation between ERalpha and ERbeta expression. 'ERalpha-negative' tumors (containing very low ERalpha mRNA levels) were associated with SBR histopathological grade III, RB1 underexpression and ERBB2 overexpression, confirming that ERalpha negativity delineates poorly differentiated tumors. The amount of ERalpha mRNA (but not that of ERbeta mRNA) increased with age and was consequently higher in postmenopausal patients' tumors. Expression of ERalpha (but not that of ERbeta) also correlated strongly with progesterone receptor (PR) and PS2 expression, suggesting that ERalpha has stronger transcriptional activity than ERbeta towards genes containing an ERE (estrogen response element) in their promoters. Interestingly, we found a negative correlation between the expression of ERbeta (but not ERalpha) and CCND1, which contains an AP1 element but not an ERE in its promoter. Taken together, these data confirm that ERalpha and ERbeta play different roles in breast cancer, partly by mediating the transcription of various genes via different types of DNA enhancer. PR and PS2 seem to be mainly ERalpha-responsive genes, whereas CCND1 may be mainly ERbeta-responsive. Our findings also underline the need for a reliable method, providing full range of quantitative values, to determine ERalpha and ERbeta status in the clinical setting.