Estrogen (E2) is involved in mediating many important functions relevant to osteoblast biology through the actions of the estrogen receptors (ER) alpha and beta. To further understand the mechanisms of ER-specific regulation, we used microarray and reverse transcription-PCR analyses of E2-treated U2OS-ERalpha or -ERbeta cells and identified retinoblastoma-binding protein 1 (RBBP1) as a major E2-regulated gene. RBBP1 is a retinoblastoma cofactor involved in the control of osteoblastic proliferation. Although RBBP1 mRNA levels rapidly increased after 2 h of E2 treatment in both U2OS-ER-expressing lines, a sustained induction was only observed in U2OS-ERalpha cells. Examination of the RBBP1 genomic sequence revealed an ER response element and a Sp1 site located within the first intron. Chromatin immunoprecipitation analyses demonstrated that E2-dependent ERalpha binding to the intron 1 enhancer region was constitutive, whereas ERbeta binding was transient, consistent with the mRNA time course. Interestingly, transient transfection and receptor mutational studies revealed that RBBP1 induction by ERalpha only requires the Sp1 site, whereas ERbeta utilizes both the Sp1 and estrogen response elements binding sites for maximal E2-dependent activation. Stable U2OS transfectants containing a deletion of the ERalpha activation function 1 (AF1) resulted in a temporal mRNA induction profile similar to that of wild type ERbeta. Further, overexpression and chromatin immunoprecipitation analyses also demonstrated that E2-dependent RBBP1 induction is SRC2-dependent for both ER isoforms. These results describe an E2-dependent, ER isoform-specific transcriptional activation of the RBBP1 gene, which in part, is explained by the differential activity of ER AF1 and enhancer element binding.