Aromatase converts testicular androgens to estrogens, which are essential for male fertility. Aromatase expression in testis occurs via transcription from promoter II, and requires the presence of a nuclear receptor half-site that binds the orphan receptor steroidogenic factor-1 [SF-1 (nuclear receptor 5A1)] to mediate basal and (in part) cAMP-induced transcription. We hypothesized that liver receptor homolog-1 (LRH-1) (nuclear receptor 5A2), a receptor closely related to SF-1, could also play a role in regulating aromatase expression in the testis. We demonstrate expression of LRH-1 in adult rat and immature mouse Leydig cells (LHR-1 > SF-1) as well as in pachytene spermatocytes and round spermatids but not in Sertoli cells, which in contrast, express high levels of SF-1. In transient transfection assays using TM3 Leydig cells and TM4 Sertoli cells, a rat promoter II luciferase reporter construct was stimulated by cotransfection of LRH-1 expression vector. Mutation analysis showed that induction by LRH-1 in TM3 and TM4 cells requires an AGGTCA motif at position -90, to which LRH-1 bound in gel shift analysis. We therefore provide evidence that LRH-1 plays an important role in the regulation of aromatase expression in Leydig cells. The colocalization of LRH-1 and aromatase to multiple testis cell types suggests that LRH-1 may have important effects on estrogen production, testis development, spermatogenesis, and testicular carcinogenesis.