Ribonuclease T1 (RNase Tl) specifically hydrolyzed the phosphodiester linkages of guanosine 3’-phosphate of single-stranded RNA and the cleavage occurs in a two-step reaction mechanism. It is considered that in the first step Glu 58 abstracts a proton from 2’-OH and His 40 or His 92 adds a proton to 0-5’ of ribose. We succeeded to express the chemically synthesized genes for RNase Tl and its several mutants at base-recognition site in E. coli and reported the structure-function relationship of this enzyme. In this paper, we changed Glu 58, His 40 and His 92 to alanine etc. and analyzed the activity of these mutant enzymes in order to clarify the role of catalytic residues. Gln 58 mutant and Ala 58 mutant still retained slight activity but both Ala 40 and Ala 92 mutants lost the activity almost completely. This result indicates that Glu 58 is not essential but His 40 and His 92 are indispensable for RNase Tl activity. We propose a new reaction mechanism, in which His 40 abstracts a proton from 2’-OH and His 92 protonates 0-5’ of ribose while Glu 58 enhances the basicity of His 40.