The enzyme aromatase converts androgens to estrogens, which have recently been postulated to be essential for testicular development and fertility. Understanding the mechanisms that regulate aromatase activity in the testis may therefore have implications for treatment of male infertility. Aromatase is encoded by the CYP19 gene, which uses multiple tissue-specific alternative promoters. In the testis, the proximal promoter PII drives aromatase expression. PII activity requires a nuclear receptor half-site, CAAGGTCA, to which two orphan receptors; SF-1 and LRH-1, have been shown to bind in vitro. The aim of this study was to investigate expression of aromatase and LRH-1 in the developing rat testis and define the ability of LRH-1 to induce aromatase expression in the testicular cells where both are expressed. We show that aromatase and LRH-1 are present throughout all stages of development of the rat testis, although the sites and levels of expression vary. The pattern of LRH-1 expression was broadly similar to that of aromatase. In adult animals higher levels of expression were observed in Leydig and germ cells. Over-expression of LRH-1 in primary rat Leydig and germ cells by adenoviral infection strongly increased endogenous aromatase mRNA levels, demonstrating the ability of LRH-1 to stimulate aromatase expression in vivo. We also observed binding of endogenous LRH-1 to the aromatase promoter II by chromatin immunoprecipitation. These data provide evidence that LRH-1 plays an important role in the regulation of testicular aromatase expression, and implicate LRH-1 as a regulator of rat spermatogenesis, in which estrogens are emerging as important mediators.