Bromoacetate reacts with histidine residues 12 and 119 at the active site of bovine pancreatic ribonuclease (RNase) much more rapidly than with free histidine. The mechanism of this facilitated alkylation was investigated by studying the dependence of the reaction on temperature and pH. RNase was treated with bromoacetate under pseudofirst-order conditions at 12, 25, 37, and 50 O C . The rate of inactivation of the enzyme showed a hyperbolic dependence on bromoacetate concentration, indicating formation of an enzyme-bromoacetate complex (K, = 41 m M at pH 5.5 and 25 "C). Two groups, one of which must be unprotonated and the other protonated, are required for carboxymethylation of RNase by bromoacetate. At 25 OC, the free enzyme exhibits macroscopic pK values of 4.7 and 6.3, and the enzymebromoacetate complex has pK values of 5.8 and 7.4. The ratio of products [N"-(carboxymethy1)histidine-1 19 RNase to N'-(carboxymethy1)histidine-12 RNase] formed in the reaction was 4.4 and was independent of temperature. Calculations based on this ratio and the microscopic pK values of histidines-1 19 and -12 determined by N M R titration suggest that the pH-independent alkylation of histidine-1 19 is about 8 times