Bis-(dichloroacetyl)-diamines (BDADs) are compounds that inhibit spermatogenesis and function as male contraceptives in many species; however, their mechanism of action has yet to be fully investigated. It has been proposed that BDADs may function via inhibition of testicular retinoic acid (RA) biosynthesis. We employed an organ culture technique and the expression of a marker for RA activity, Stra8 (stimulated by retinoic acid gene 8), to investigate if the BDAD WIN 18,446 inhibited the biosynthesis of RA from retinol (ROL) in neonatal and adult murine testis and in the embryonic murine gonad. After culturing either whole testes or germ cells isolated from mice at 2 days postpartum (dpp) with WIN 18,446 or with WIN 18,446 plus ROL, Stra8 expression was suppressed, demonstrating that WIN 18,446 inhibited the conversion of ROL to RA in both systems. We also utilized a transgenic mouse containing an RA-responsive LacZ reporter gene to demonstrate limited RA induction of LacZ expression in 2-dpp testes cultured with WIN 18,446 plus ROL. The expression of Stra8 was downregulated in adult mouse testis tubules cultured with WIN 18,446 when compared to tubules cultured with the vehicle control. WIN 18,446 also inhibited the conversion of ROL to RA in embryonic ovaries and testes cultured for 48 h. These murine results provide critical insights regarding how the BDADs can inhibit spermatogenesis by blocking the ability of vitamin A to drive germ cell development. In addition, these techniques will be useful for screening novel inhibitors of RA biosynthesis as potential male contraceptives.