Four spin-labeled analogs of adenosylcobalamin have been synthesized to aid in the detection and identification of radical intermediates in the adenosylcobalamin-dependent enzymatic reactions and to serve as probes of the coenzyme, substrate, and effector binding sites of the protein. Three isomers of adenosylcobalamin, in which one of the propionamide side chains (b, d, or e) was hydrolyzed, and adenosylepicobalamin e-carboxylic acid were reacted with 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to yield the spin-labeled adenosylcorrinoids. These spin-labeled derivatives of adenosylcobalamin function as coenzymes and/or inhibitors of dioldehydrase from Klebsiella pneumoniae and of ribonucleotide reductase from Corynebacterium nephridii. Electron spin resonance has been used to monitor the photolytic cleavage of the carbon-cobalt bond of these analogs.