S-Adenosyl-L-methionine (AdoMet), S-adenosyl-L-homocysteine (L-AdoHcy), and related ribonucleosides have been oxidized with periodic acid to the corresponding 2',3'-dialdehydes. Both AdoMet dialdehyde and L-AdoHcy dialdehyde were observed to rapidly and irreversibly inactivate histamine N-methyltransferase (HMT). Equally active as an irreversible inhibitor was S-adenosyl-D-homocysteine dialdehyde (D-AdoHcy dialdehyde), which is consistent with the known affinity of HMT for S-adenosyl-D-homocysteine (D-AdoHcy). Other analogues of AdoHcy dialdehyde (S-adenosyl-L-cysteine dialdehyde, S-adenosyl-L-homocysteine sulfoxide dialdehyde, and adenosine dialdehyde) also produced irreversible inactivation of HMT, but at predictably slower rates. The corresponding acyclic 2',3'-ribonucleosides, which were obtained by NaBH4 reduction of the ribonucleosides dialdehydes, were found to be very weak, reversible inhibitors of HMT. Kinetic analysis of the inactivation of HMT produced by L-AdoHcy dialdehyde, AdoMet dialdehyde, and D-AdoHcy dialdehyde suggested mechanisms involving the formation of dissociable enzyme-inhibitor complexes prior to irreversible inactivation. Studies using L-[2,8-3H] AdoHcy dialdehyde revealed that incorporation of radioactivity into HMT closely paralleled the loss of enzyme activity. The results of these studies indicate that L-AdoHcy dialdehyde, D-AdoHcy dialdehyde, and AdoMet dialdehyde are affinity labeling reagents for HMT.