Abstract d-Aspartate increases human chorionic gonadotropin-induced testosterone production in purified rat Leydig cells. l-Aspartate, d-, l-glutamate or d-, l-asparagine could not substitute for d-aspartate and this effect was independent of glutamate receptor activation. Testosterone production was enhanced only in cells cultured with d-aspartate for more than 3 h. The increased production of testosterone was well correlated with the amounts of d-aspartate incorporated into the Leydig cells, and l-cysteine sulfinic acid, an inhibitor of d-aspartate uptake, suppressed both testosterone production and intracellular d-aspartate levels. d-Aspartate therefore is presumably taken up into cells to increase steroidogenesis. Intracellular d-aspartate probably acts on cholesterol translocation into the inner mitochondrial membrane, the rate-limiting process in steroidogenesis.