The photolysis of adenosylcobalamin (coenzyme B12) results in homolytic cleavage of the Co-C5' bond, forming cob(II)alamin and the 5'-deoxyadenosyl radical. In the presence of molecular oxygen, it has been proposed that the primary reaction is interception of the 5'-deoxyadenosyl radical by O2 to form adenosine-5'-aldehyde as the product (Hogenkamp, H. P. C., Ladd, J. N., and Barker, H. A. (1962) J. Biol. Chem. 237, 1950-1952). 5'-Peroxyadenosine is here found to be the initial nucleoside product of this reaction and found to decompose to adenosine-5'-aldehyde. Evidence indicates that 5'-peroxyadenosine arises from the hydrolysis of 5'-peroxyadenosylcobalamin, with the formation of cob(III)alamin. 5'-Peroxyadenosine undergoes further decomposition to adenosine-5'-aldehyde as the major final product of aerobic photolysis as well as to adenosine and adenine as minor products. In a cobalamin-dependent process, 5'-peroxyadenosine becomes re-ligated to cob(III)alamin to form 5'-peroxyadenosylcobalamin, which quickly decomposes to adenosine-5'-aldehyde and cob(III)alamin. This is supported by spectrophotometric observations of both rapidly photolyzed adenosylcobalamin and the reaction of cob(III)alamin with excess 5'-peroxyadenosine. 5'-Peroxyadenosine also slowly undergoes cobalamin-independent decomposition to adenosine-5'-aldehyde and the minor products adenosine and adenine. The present study provides a detailed description of the products initially formed when aqueous, homolytically cleaved adenosylcobalamin reacts with molecular oxygen and provides a detailed description of the behavior of those products subsequent to photolysis.