The proliferation and differentiation of Sertoli cells is regulated by follicle-stimulating hormone (FSH). The molecular events following FSH stimulation are only partially known. To investigate FSH action in Sertoli cells, we established two novel FSH-responsive mouse Sertoli-cell-derived lines expressing human wild-type (WT) FSH receptor (FSHR) or overexpressing mutated (Asp567Gly) constitutively active FSHR (MUT). Gene expression profiling with commercially available cDNA arrays, including 588 mouse genes, revealed 146 genes expressed in both cell lines. Compared with the expression pattern of WT cells, 20 genes were identified as being either up- or down-regulated (>two-fold) in the MUT cells. We observed a strong differential expression of factors involved in cellular proliferation, e.g. cyclin D2 (repressed to nearly undetectable levels), proliferating cell nuclear antigen (2.5-fold repression) and Eps-8 (six-fold repression), and in genes involved in cellular differentiation, e.g. cytokeratin-18 (13-fold induction). The cDNA array results for six representative genes were confirmed by Northern blotting, which also included the parental SK-11 cell line devoid of FSHR expression. We found no further acute FSH- or forskolin-induced change in expression levels after 3-h stimulations, suggesting that the observed differences between the two cell lines is a consequence of mild, chronically increased, cAMP production in MUT cells. These results provide a platform for the further investigation of selected candidate genes in primary cultures and/or in vivo.