Active-site histidine residues of bovine seminal RNase have been found to react with bromoacetic acid and with 2'(3')-O-bromoacetyluridine (BrAcUrd) at a much faster rate than free histidine. The former reagent reacts preferentially at the pros-N of His119, the latter is specific for the tele-N of His12. Alkylation with bromoacetic acid is mutually exclusive for either His119 or His12 and takes place predominantly at His119, while with BrAcUrd alkylation was found to be selective for His12. These results are very similar to those obtained with the same reagents on RNase A, confirming that seminal and pancreatic ribonucleases have similar geometries at their active sites. On the other hand, the kinetics of reaction of bromoacetyluridine with seminal RNase reveal a 'half-of-the sites' reactivity of the enzyme for this reagent, which is found to discriminate between the two structurally identical active sites of the dimeric enzyme.