Advances in photochemical generation of “caged” biochemical intermediates ( 1) have made it possible to examine kinetic events that are too fast or inappropriate for stopped-flow methods. We have done experiments to test whether the chemistry of photochemical generation of organic phosphates can be used in the presence of spinlabeled macromolecules. It was a concern that free-radical intermediates generated in the photolysis might react with the nitroxide groups. Specifically, we have examined the known conformational change of spin-labeled phosphorylase b when it binds AMP ( 2). The effector, AMP, was generated photochemically from its 1 ( 2-nitrophenyl ) ethyl ester. For the test, the photolysis was done with a mercury arc lamp. The results suggest that it should be feasible to conduct experiments in the time-resolved mode using EPR detection with spin labels and laser-induced photolysis of caged intermediates to initiate events. Phosphorylase b is a dimer that exhibits cooperativity between subunits (3). The protein can be labeled with an iodoacetamide spin label at cysteine-3 17 (4). The factors that contribute to altered EPR signals when spin-labeled phosphorylase b from rabbit muscle interacts with effecters have been examined in detail (2). Phosphorylase b is activated when AMP binds to the activator site and the EPR signal of the spinlabeled protein changes on AMP binding. In addition, there is a different nucleoside inhibitor site to which adenine, adenosine, and other inhibitors bind. Pyridoxal 5’phosphate serves as a cofactor. Caged AMP was prepared (5) by DCC coupling of I-(2-nitrophenyl)ethanol to pyridinium AMP in dry pyridine. After complete removal of pyridine, caged AMP was separated from starting materials on a DEAE-A25 Sephadex column (bicarbonate form, pH 7.0, 0 to 0.26 M triethylammonium bicarbonate linear gradient). Thinlayer chromatography on silica gel plates in a solvent system of isopropanol, ammonium hydroxide, water (7, 2, 1 v/v) gave a spot with Rf 0.82 which gave, after photolysis, a spot of Rf 0.33, identical to that of AMP. Microanalysis was consistent with the formula of caged AMP * 2.5 H20 and inconsistent with the formulas of starting materials. Phosphorylase b was prepared with modifications of the procedure of Fischer