Transcription of the rat P450c17 gene in Leydig cells requires steroidogenic factor-1 (SF-1, NR5A1), nerve growth factor-inducible protein B (NGFI-B, nurr77), COUP-TF, and SET. The -447/-419 region of this promoter contains two binding sites for orphan nuclear receptors that are required for activation by SF-1, NGF-IB, and cAMP. We identified a novel factor, Steroidogenic Factor-Inducer of Transcription-2 (StF-IT-2) that binds to this -447/-419 region. We have now purified StF-IT-2 from mouse Leydig MA-10 cells and identified it by mass spectrometry as translin, a 27 kDa protein that exerts many functions. By itself, translin cannot activate a P450c17-promter/reporter construct in HeLa cells; however, translin increased SF-1-stimulated transcription 2-fold, indicating cooperativity between SF-1 and translin. Mutation of both SF-1 binding sites in the -447/-419 sequence eliminated activation by SF-1 and translin. Translin did not augment SF-1-stimulated transcription from all SF-1-responsive elements, suggesting that the activation is specific for the sequence of the SF-1 response element. Gel shift analysis of double and single stranded DNA showed that translin binds to single stranded DNA, but its transcriptional activation is independent of DNA binding. The hinge region of SF-1 is necessary for activation by translin; deletion of hinge amino acids 170 - 225 in SF-1 eliminates translin's ability to augment SF-1-dependent transcription. A translin-like protein, called TRAX, can substitute for a translin moiety; translin homomers and translin/TRAX heteromers activated SF-1-stimulated transcription equally. Thus we have identified a new factor that works together with SF-1 to augment gene transcription in a DNA-specific fashion.