In the present study, we investigated the signaling pathway involved in luteinizing hormone (LH)-mediated regulation of testicular CYP1B1 in mouse MA-10 and rat R2C Leydig cells. CYP1B1 mRNA and protein levels were measured in MA-10 and R2C cells treated with LH and protein kinase activators or inhibitors. Treatment with LH or 8-bromo-cAMP, a protein kinase A (PRKA) activator, increased CYP1B1 expression and PRKA activity in a concentration-dependent manner in both cell lines, albeit to different extents. Treatment with 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphorothioate, Rp-isomer, a PRKA inhibitor, decreased basal CYP1B1 expression and attenuated LH-elicited increases in CYP1B1 mRNA and protein levels and PRKA activity. In contrast, treatment with a protein kinase G activator or an inhibitor of protein kinase C had no effect on basal or LH-induced CYP1B1 expression in MA-10 or R2C cells. Collectively, the results identify PRKA as the major signaling pathway involved in the LH-mediated regulation of testicular CYP1B1 expression in Leydig tumor cells.