Steroid biosynthesis activated by pituitary tropic hormones is known to be acutely regulated by cAMP acting via Protein kinase A. Because the mitochondrial benzodiazepine receptor (MBR) has been suggested to play a role in the activation of steroidogenesis, the present study investigates whether various protein kinases phosphorylate MBR. In rat and bovine adrenal mitochondrial preparations Protein kinase A, but not other purified protein kinases, was found to phosphorylate the 18 kDa MBR protein. In digitonin-permeabilized MA-10 Leydig tumor cells incubated with [gamma-32P]ATP, phosphorylation of MBR was detectable during treatment of the cells with dibutyryl cAMP. In conclusion, these data show that the MBR protein is an in vitro and in situ substrate of Protein kinase A, but the role of this phosphorylation in the regulation of steroidogenesis remains to be established.