Femtosecond transient absorption spectroscopy has been used to investigate the photolysis of coenzyme B-12 (5＇-deoxyadenoSylcobalamin). Transient kinetic measurements obtained at wavelengths between 400 and 633 nm were analyzed globally to obtain time constants. Transient spectral data obtained for time delays between 5 ps and 9 ns were analyzed by matrix decomposition to identify distinct spectral components present in the data. Photoexcitation results in homolysis of the carbon-cobalt bond forming a singlet radical pair on a picosecond time scale. The subsequent spectral changes probe conformational relaxation and geminate recombination. Analysis of the spectral data suggests that 76 +/- 4% of the geminate radical pairs recombine, resulting in a quantum yield of 0.24 +/- 0.04 for the formation of solvent separated radicals, in good agreement with literature values of 0.20 +/- 0.03 and 0.23 +/- 0.04 [Chen, E.; Chance, M. R. Biochemistry 1993, 32, 1480-1487]. The geminate recombination of the adenosyl radical with cob(II)alamin occurs biphasically with exponential time constants of 150 +/- 20 ps and 0.5 +/- 0.2 ns. The effective recombination rate from a single-exponential fit to the data is (0.250 ns)(-1) = 4 ns(-1).