Two adenosine residues, universally conserved among group I introns, are located in the L4 egion of the catalytic core. Base-substitution mutations in these adenosines resulted in diminished in vitro self-splicing activity of the Tetrahymena group I intron, more severely for double than for single mutations. The defect caused by the mutation of the L4 adenosines was manifest at the first step of splicing (cleavage of the 5' splice site by a guanosine molecule), and could be overcome by increasing the magnesium ion concentration of the reaction buffer. In contrast, a related activity of the group I intron, specific hydrolysis of the 3' splice site, was virtually unaffected by the L4 adenosine mutations; this reaction must require an active conformation of the catalytic site of the ribozyme and also depends on the recognition of guanosine (the guanosine residue that precedes the 3' splice site in all group I introns). These results suggest that the role of the L4 adenosines is limited to improving the reactivity of the 5' splice site during splicing. We also found that mutations that eliminate the 5' splice site-bearing P1 stem and the P10 stem have little or no effect on specific hydrolysis of the 3' splice site.