The changing relationship between stimuli and responses after prolonged receptor stimulation is a general feature of hormonal signaling systems, termed desensitization. This phenomenon has been best exemplified in the covalent modification of the G protein-linked catecholamine receptors. However, other components within this signaling pathway can be involved in desensitization. Here we present evidence that desensitization occurs at the level of the effector enzyme itself through phosphorylation. Type V adenylyl cyclase (AC) is the major isoform expressed in the heart. Using purified enzymes, we demonstrate that protein kinase A (PKA) directly phosphorylates and thereby inhibits type V AC catalytic activity. This inhibition was negated in the presence of PKA inhibitor. Analysis of enzyme kinetics revealed that this inhibition was due to a decrease in the catalytic rate, not to a decrease in the affinity for the substrate ATP. Our results indicate that AC catalytic activity can be regulated through PKA-mediated phosphorylation, suggesting another mechanism of desensitization for receptor pathways which signal via increases in intracellular cAMP.