In order to study the interaction between B_<12> derivatives and SH groups of protein, the effect of B_<12> derivatives on the activity of thiol enzymes, papain and yeast alcohol dehydrogenase (YADH), was investigated. Both papain (7×10^<-2>mM) and YADH (1.4×10^<-3>mM) were inhibited by the incubation with 5.0×10^<-2>mM to 1.0×10^<-2>mM of aquocobalamin (OH-B_<12>) or cyanocobinamide. The inhibition of these thiol enzymes with OH-B_<12> was increased rapidly at pH greater than 6.0. Under anaerobic conditions, papain was scarecely inhibited, while YADH was inhibited as much as under aerobic conditions. The inhibitory effect of OH-B_<12> on YADH was merkedly decreased by the addition of NAD or NADH in the reaction medium. The inhibited papain was reactivated by KCN treatment. On the other hand, the inhibited YADH was not significantly reactivated by cysteine. The SH groups of the inhibited enzymes were found to decrease in comparison with those of the native enzymes.