The radical intermediate present during steady-state turnover of substrate aminoethanol by ethanolamine deaminase from Salmonella typhimurium has been characterized by using X-band electron paramagnetic resonance (EPR) spectroscopy. The radical intermediate was prepared by cryotrapping enzyme, aminoethanol substrate, and vitamin B-12 coenzyme (adenosylcob(III)alamin) immediately following mixing. Natural abundance, 1,1,2,2-H-2(4)-, 2-C-13-, and 1,2-C-13(2)-aminoethanol were used as substrates. The EPR spectrum obtained for natural abundance aminoethanol shows a broad feature at approximately g = 2.3 that arises from Co-II in cob(LI)alamin, and a feature from an organic radical that has an absorption maximum at g = 2.02 and a line width of 10.8 mT. The EPR line shape is characteristic of a relatively weakly electron spin-coupled Co-II-organic radical system. The EPR line shapes for the H-2- and C-13-labeled substrates were narrowed and broadened by 0.7 and 2.4 mT, respectively, demonstrating that the radical is substrate-based. The comparable line widths of the 2-C-13- and 1,2-C-13-labeled radicals show that the unpaired spin density is localized primarily at the C-2 carbon atom. This identifies the radical intermediate as a rearranged substrate radical, or product radical. The results are consistent with either the 1-aminoethanol-2-yl radical or the ethanal-2-yl radical, which have been proposed as intermediates in, respectively, the amine migration and amine elimination mechanisms of rearrangement. A qualitative reaction free energy profile for the Con-radical pair intermediate states on the enzyme is constructed, based on the EPR results and previous isotope exchange and kinetic isotope effect studies. The results and analysis reveal that a product radical trap strategy contributes to the stabilization of the radical pair state, which enhances catalytic performance of ethanolamine deaminase.