The highly conserved P7 region is generally believed to act as a major portion of the catalytic site in the Group I intron ribozyme. However, its functions have not been elucidated except for the fact that it specifically binds a cofactor guanosine required for self-splicing reaction. We attempted an in vitro selection experiment to determine the sequence requirements of this region in the mechanism of catalysis by using the Tetrahymena ribozyme. We found that the selected active clones have the secondary structure similar to that of the wild type with few exceptions. However, their primary sequences were not conserved except G264 and C311 that are the major elements of the binding site for the guanosine. Our results suggest that the unique secondary structure of the P7 region is a primary requisite for the catalytic function of this class of ribozymes.