A combination of five thermostabilizing mutations, Gly23fiAla, His62fiPro, Val74fiLeu, Lys95fiGly, and Asp134fiHis, has been shown to additively enhance the thermostability of Escherichia coliRN-ase HI [Akasako A, Haruki M, Oobatake M & Kanaya S (1995)Biochem-istry 34,8115–8122]. In this study, we determined the crystal structure of the protein with these mutations (5H-RNase HI) to analyze the effects of the mutations on the structure in detail.