The heart is highly energy-dependent with most of its energy provided by mitochondria. Mitochondria also play a role in many essential cellular processes including metabolite synthesis, redox balance, calcium homeostasis and cell death. Therefore, maintaining a functional population of mitochondria is critical for cardiac function and identification of novel regulatory mechanisms. Recently, a soluble adenylyl cyclase (sAC) has been revealed to serve as a local source of the second messenger cAMP in the mitochondrial matrix in response to bicarbonate and calcium. PDE2A, a cGMP-stimulated cAMP-hydrolyzing phosphodiesterase (PDE), was also shown to be expressed in the mitochondrial matrix of liver and brain. To gain new insights into the control of mitochondrial pool of cAMP, we investigated the role of various isoforms of PDEs in isolated cardiac mitochondria. Western blot analysis of PDE protein expression revealed positive bands for PDE2A, 3A, 4A and 4B subtypes in cardiac mitochondria. Basal cAMP-degrading enzymatic activities were determined by radioenzymatic assay in cardiac mitochondrial lysates with 1μm cAMP as substrate. PDE2 represented the largest mitochondrial cAMP PDE activity (35% of total) and its activity was enhanced ~2-fold by 5μM cGMP and was inhibited by the PDE2 inhibitor, Bay 60-7550 (100nm). PDE3 and PDE4 activities represented, respectively, 30% and 25% of total, and they were inhibited by their respective inhibitors, cilostamide (1μm) and Ro 20- 1724 (10 μM). Accordingly, measurements by ELISA of mitochondrial cAMP levels confirmed that inhibition of PDE2, 3 and 4 families leads to an increase in cAMP level. In conclusion, at least three PDE families and four PDE subtypes are located in the cardiac mitochondrial matrix, participating to a local signaling pathway with sAC to control cAMP level. Our findings unravel a cAMP signaling cascade in cardiac mitochondria which may have implications for the metabolic control of cardiac function.