Methylation interference experiments reveal bases involved in three different catalytic functions of the T4-phage derived sunY self-splicing intron. RNA molecules methylated at the N-7 position of the guanine at the cofactor binding site are inactive in cofactor-dependent splicing and 3 splice-site hydrolysis. In contrast, 5 splice-site hydrolysis occurs despite methylation at this position. Specific adenines that have been implicated in docking of the P1 stem to the catalytic core are shown to be important for cofactor-dependent splicing and essential for 5 splice-site hydrolysis. Similarly, methylation of bases in the P9.0 stem, as well as C56 in J5/4, interferes with 3 splice-site hydrolysis and with the splicing reaction. All of the bases identified as important for the overall splicing reaction are also identified as essential for either the 5 or 3 splice-site hydrolysis reactions, and vice versa. It is inferred that the bases implicated in 5 and 3 splice-site hydrolysis are involved in specific interactions of the 5 and 3 splice site, respectively, with the catalytic core.