The orphan nuclear receptor steroidogenic factor-1 (SF-1) is involved in the transcriptional regulation of all the steroid hydroxylase genes, and also regulates the transcription of the genes for Müllerian Inhibitory substance (MIS), alpha subunit of glycoprotein hormone, LHbeta, oxytocin, GnRH receptor, ACTH receptor, prolactin receptor, DAX-1, and steroidogenic acute regulatory protein. Other members of the nuclear receptor gene family, including steroid hormone, thyroid hormone, retinoic acid, PPAR, and vitamin D receptors must bind ligand to activate transcription, but SF-1 has been considered to be an orphan nuclear receptor because, when identified, it had no known ligand. A recent publication suggested that transcriptional regulation by SF-1, expressed in a non-steroidogenic CV-1 cells, could be activated by oxysterols suggesting that these compounds could serve as natural ligands for SF-1. We now demonstrate that 25-hydroxycholesterol, either added exogenously or synthesized endogenously in steroidogenic mouse Leydig MA-10 cells, did not act as a ligand for SF-1, as it did not increase transcription from six different SF-1-dependent DNA sequences. Furthermore, the abundance of these oxysterols in MA-10 cells was much less than concentrations needed for activation of SF-1 in CV-1 cells, indicating that SF-1 is not constitutively bound by ligand in MA-10 cells. Thus, in steroidogenic cells, transcriptional regulation of the steroid hydroxylase genes by SF-1 does not depend upon the presence of 25-hydroxycholesterol, and is not modified by its presence.