8-Azidoadenosine 3',5'-monophosphate (8-N3-cAMP) containing 32P has been used as a photoaffinity label specific for the adenosine 3',5'-monophosphate (cAMP) binding site(s) present in a partially purified preparation of soluble protein kinase from bovine brain. 8-N3-cAMP and cAMP were found to compete for the same binding site(s) in this preparation, as determined by a standard filter assay. When this protein preparation was equilibrated with [32P]-8-N3-cAMP, and then irradiated at 253.7 nm, the incorporation of radioactivity was predominantly into a protein with an apparent molecular weight of 49,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. This labeled protein comigrated in the gel with the only protein which is endogenously phosphorylated by [gamma-32P]ATP, a protein which has been shown to be the regulatory subunit of the protein kinase (H. Maeno, P. L. Reyes, T. Ueda, S. A. Rudolph, and P. Greengard (1974), Arch. Biochem. Biophys. 164, 551). The incorporation of [32P]-8-N3-cAMP into this protein was half-maximal at a concentration of 7 x 10(-8) M. In accordance with a proposed mechanism involving the formation of a highly reactive nitrene intermediate upon irradiation of the azide, the incorporation of radioactivity into protein was maximal within 10 min of irradiation, and was almost eliminated by preirradiation of the photolabile ligand. Moreover, this incorporation was virtually abolished by a 50-fold excess of cAMP, but not by AMP, ADP, ATP, or adenosine. We suggest that 8-N3-cAMP may prove to be a useful molecular probe of the cAMP-binding site in receptor proteins and report its use in conjunction with sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a highly sensitive and selective radiochemical marker for cAMP-binding proteins.