Leydig cells produce testosterone in the testes under the pulsatile control of pituitary luteinizing hormone (LH). cAMP is the intracellular messenger for LH action on steroidogenesis, and pharmacological evidence indicates that the response to LH can be modulated by cyclic nucleotide phosphodiesterases (PDEs). However the types and roles of the PDEs present in Leydig cells have not been fully defined. We report here that PDE8A is expressed in Leydig cells, and using PDE8A knockout mice we provide evidence that PDE8A is a key regulator of LH signaling and steroidogenesis. A 4-fold increase in the sensitivity to LH for testosterone production was detected in Leydig cells isolated from PDE8A knockout mice. In Leydig cells from wild-type mice, 3-isobutyl-1-methylxanthine, a compound that inhibits all cAMP PDEs except PDE8A, elicited only a small increase in the sensitivity of testosterone production to LH. However, in the PDE8-null mice, the effect of this inhibitor is much more pronounced. These observations indicate that PDE8A and at least one other PDE control the same or a complementary pool of cAMP that mediates LH-regulated steroidogenesis. Overall, these results suggest that pharmacological manipulation of PDE8A, alone or in combination with other PDEs present in Leydig cells, may be exploited to modulate testosterone synthesis and possibly to treat various conditions where the local levels of this androgen need to be altered.