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.cAMP ͉ testosterone ͉ PDE8A ͉ testis ͉ steroidogenesis T he second messenger cAMP plays important roles in mediating the biological effects of a wide variety of first messengers. Increases in intracellular cAMP lead to activation of cAMP-dependent protein kinases, guanine nucleotide exchange factors, and cyclic nucleotide-gated channels, which in turn can regulate the activity of other signaling and metabolic pathways (1). cAMP signaling pathways are controlled through regulation of the synthesis of cAMP by adenylyl cyclases and degradation by phosphodiesterases (PDEs) (1, 2). The cyclic nucleotidePDEs are now recognized to form a superfamily of 11 different, but homologous, gene-families that all contain highly homologous catalytic domains near their C termini (3). PDE-catalyzed cyclic nucleotide degradation provides an important mechanism for regulating signaling. Indeed, the PDE component of cAMP pathways ensures the proper intensity and spatiotemporal distribution of the signal (4, 5), as illustrated by many studies on different endocrine tissues (6-8).Leydig cells are interstitial cells located adjacent to the seminiferous tubules in the testes. The best-established function of Leydig cells is to produce the androgen, testosterone, under the pulsatile control of pituitary luteinizing hormone (LH) (9). It has been demonstrated that cAMP is the major intracellular messenger for LH action on steroidogenesis and that most, if not all, of the signaling action of cAMP is due to cAMP-dependent protein kinase (PKA)-mediated effects on proteins regulating the steroid biosynthetic pathway (9...