The effect of photo-oxidation and carboxymethylation on the activity of RNAse Pch1 has been studied. Photoinactivation in the presence of rose bengal results in a selective oxidation of two histidine residues. The process is inhibited by the nucleotide substrate analogs. This suggests that one or two imidazole groups may be localized in the active site of RNAse Pch1. The pH dependence of the enzyme inactivation by bromoacetic acid is indicative of the contribution of a functional group with pKa 4,0, presumably of a beta- or gamma-carboxyl group of dicarbonic amino acid. The reaction is inhibited by the substrate analogs 2'(3')-GMP and 2'(3')-AMP. The data on the similarity of active sites in several guanyloribonucleases are discussed.