Ultraviolet irradiation of creatine kinase (adenosine 5'-triphosphate: creatine-N-phosphotransferase, EC 18.104.22.168) in the presence of gamma-(p-azidoanilide)-[14C]-ATP results in a complete enzyme inactivation and covalent binding of two moles of analog per mole of enzyme. MgADP strongly protects the enzyme against inactivation. However, in the presence of MgADP the covalent binding of 0.9 moles of analog per mole of enzyme occurs without the enzyme inactivation. The rate of enzyme inactivation and covalent modification decreases in the presence of MgATP and creatine. These results indicate that gamma-(p-azidoanilide)-ATP is an affinity label for the active site of creatine kinase.