In this study, an estrogen receptor (ER) alpha-expressing T47D cell line containing an inducible tet-off FLAG-ERbeta was used to examine the influence of ERbeta on ERalpha activity. Real-time PCR analysis of mRNA levels of two well-studied estrogen-responsive genes, pS2 and progesterone receptor (PR), showed that the expression levels of both genes were reduced in the presence of ERbeta. Chromatin immunoprecipitation assays showed that the 17beta-estradiol (E2)-induced recruitment patterns to the pS2 and PR promoters were similar for both ERalpha and ERbeta. ERbeta expression did not significantly influence the kinetic recruitment profile of ERalpha to the pS2 promoter, but it was evident that ERalpha occupancy at the PR promoter was reduced. The E2-induced recruitment of c-Fos to a 12-O-tetradecanoylphorbol-13-acetate response element site in the PR promoter was significantly reduced in the presence of ERbeta, whereas only a slight reduction in the recruitment of c-Fos to the pS2 promoter was observed. ERbeta expression resulted in a significant reduction in the E2-induced expression of c-Fos mRNA. The recruitment pattern of c-Jun was also altered by ERbeta, although the expression levels of c-Jun were not. Expression of ERbeta caused a further 30-50% decrease of the E2-induced reduction in ERalpha protein after 3 h of E2 treatment, showing that ERbeta influences ERalpha protein levels. The altered recruitment of the activating protein-1 complex, combined with the reduction in ERalpha protein levels, may partly explain the antagonistic effect of ERbeta on ERalpha-mediated transcription.