Estrogen receptors alpha and beta (ERalpha and ERbeta) are expressed in the cerebellum throughout development and in the adult suggesting an important role of 17-beta-estradiol (E2) in this brain structure. In the present study, we have characterized the functionality of estrogen receptors (ERs) expressed in the immature cerebellar granule cell line E(t)C.1 by transfecting such cells with a luciferase reporter gene (ERE-Luc) coupled to an estrogen response element promoter. The induction of luciferase activity in E(t)C.1 cells by E2 and ER-subtype selective agonists was compared in normal cells and in cells overexpressing human ERalpha or ERbeta (hERalpha or hERbeta). E2-mediated transcription of the reporter gene was blocked by the ER antagonist ICI 182,780 (ICI), demonstrating the presence of functional native ERs. The selective agonist for ERalpha (PPT) showed a reduced response in luciferase induction compared to E2. Moreover, the ERbeta agonist (DPN) was unable to induce luciferase activity. E2-induced ERE-Luc transcription was not increased by overexpression of hERalpha. In contrast, hERbeta overexpression reduced the efficacy of E2 and abolished ERalpha-selective agonist activity. The ERbeta-specific agonist did not induce gene reporter activity unless hERbeta was overexpressed in the cells, suggesting that the endogenous ERbeta in E(t)C.1 cells is transcriptionally inactive. ICI inhibition of E2 responses was not affected by overexpression of the human ERs. The data suggest that ERalpha plays a predominant role in E2-mediated transcription in E(t)C.1 cells. Our data are discussed in view of other reports alluding to the complexity and cell-type specificity of E2-mediated transcription.