Analysis of luteinizing hormone (LH) beta subunit promoters from a broad range of species including teleosts and humans revealed strict conservation of a sequence homologous to the steroidogenic factor-1 (SF-1) regulatory element of cytochrome P-450 steroid hydroxylase genes. Interaction between SF-1 and this putative response element in the bovine LH beta promoter was confirmed by electrophoretic mobility shift assays. Furthermore, cotransfection of alpha T3-1 cells with an expression vector encoding SF-1 induced binding site-dependent transcription from the bovine LH beta promoter. Physiological significance of the LH beta SF-1 consensus sequence was established using transgenic mice containing either the wild type bovine promoter or a promoter with a site-specific mutation of this site. Mutation of the SF-1 binding site nearly eliminated promoter activity, and the mutant transgene remained inactive following induction of gonadotropin-releasing hormone accomplished by castrating male and female mice. Thus, increases of gonadotropin-releasing hormone within a physiological range did not compensate for the loss of the SF-1 binding site. Together, these findings indicate that the SF-1 binding site is a key regulator of LH beta promoter activity in vivo and implicate SF-1 as at least one of the transcription factors that acts through this site.