FSH is a heterodimeric glycoprotein hormone secreted from the gonadotrope cell population of the anterior pituitary. Despite its crucial role in mammalian reproduction, very little is known about regulation of the FSH β-subunit gene at the molecular level. In this report, we examine the basis for cell-specific expression of FSHβ using the mouse LβT2 and αT3-1 gonadotrope-derived cell lines. Characterization of the hormonal content of LβT2 and αT3-1 cells at the protein level classifies these cells as relatively mature and immature gonadotropes, respectively. We studied LβT2 cell-specific expression of FSHβ using 398 bp of the mouse FSHβ regulatory region linked to a luciferase reporter gene in transient transfection assays. This mouse FSHβ promoter can direct reporter gene expression specifically to LβT2 cells when compared with other pituitary- and non-pituitary-derived cell lines, including αT3-1 cells. Furthermore, it is induced by activin, and interruption of the autocrine activin loop in LβT2 cells by the addition of follistatin reduces its expression. Truncation analysis indicates that several regions of the promoter are involved in this specificity and that these can be dissociated from activin regulation. We identify binding sites for the orphan nuclear receptor steroidogenic factor-1 and the heterotrimeric transcription factor nuclear factor Y and show that these elements functionally interact to regulate FSHβ gene expression in an LβT2 cell-specific manner. Moreover, steroidogenic factor-1 and nuclear factor Y are shown to physically interact with each other. This study is the first to demonstrate the presence of basal FSHβ protein in LβT2 cells and to identify specific elements within the FSHβ promoter that contribute to basal and cell-specific expression of the gene.