Here we describe the generation of an inducible Cre transgenic line allowing conditional mutagenesis in ovarian granulosa cells. We have expressed the tamoxifen inducible CreER(T)² fusion protein from a Bacterial Artificial Chromosome (BAC) containing the regulatory elements of the hydroxysteroid (17-beta) dehydrogenase 1 (Hsd17b1) gene. Hsd17b1-iCreER(T)² transgenic mice express the iCreER(T)² fusion protein exclusively in ovarian granulosa cells. Recombination analysis at the genomic DNA level using mice with "floxed" Stat3 alleles showed no Cre activity in absence of tamoxifen whereas tamoxifen treatment induced Cre activity solely in the ovaries. Further characterization of Hsd17b1-iCreER(T)² mice using a Cre reporter line demonstrated that Cre-mediated recombination was restricted to ovarian granulosa cells. Therefore, Hsd17b1-iCreER(T)² mice should be a useful tool to analyze the gene functions in ovarian granulosa cells.