Adult Leydig cell steroidogenesis is dependent on luteinising hormone (LH) but fetal Leydig cells can function independently of gonadotropin-stimulation. To identify factors which may be involved in regulation of fetal Leydig cells EST libraries from fetal and adult testes were compared and fetal-specific genes identified. The adrenocorticotropic hormone (ACTH) receptor (melanocortin type 2 receptor (Mc2r)) was identified within this fetal-specific group. Subsequent real-time PCR studies confirmed that Mc2r was expressed in the fetal testis at 100-fold higher levels than in the adult testis. Incubation of fetal or neonatal testes with ACTH in vitro stimulated testosterone production more than 10-fold although ACTH had no effect on testes from animals aged 20 days or older. The steroidogenic response of fetal and neonatal testes to a maximally-stimulating dose of hCG was similar to the response shown to ACTH. The ED₅₀ for ACTH, measured in isolated fetal and neonatal testicular cells, was 5 x 10^-10M and the lowest dose of ACTH eliciting a response was 2 x 10^-11M. Circulating ACTH levels in fetal mice were around 8 x 10^-11M. Neither -melanocyte-stimulating hormone (-MSH) or (-MSH, had any effect on androgen production in vitro at any age. Fetal testosterone levels were normal in mice which lack circulating ACTH (POMC-null) indicating that ACTH is not essential for fetal Leydig cell function. Results show that both LH and ACTH can regulate testicular steroidogenesis during fetal development in the mouse and suggest that fetal Leydig cells, but not adult Leydig cells, are sensitive to ACTH-stimulation.