In order to determine base requirements in loops 2 and 4 of the hairpin ribozyme, a comprehensive mutational analysis of the wild type sequence was done. Each base position in these two loops was mutated to contain each of the three non-wild type bases, and the effects of these mutations were analyzed using cis-cleavage assays. The method of data analysis allowed for the determination of self-cleavage rates as well as the fraction of transcripts produced which were uncleavable. Three positions in loop 2 (A22, A23, and C25) and one position in loop 4 (A38) resulted in no detectable self-cleavage when mutated to any of the non-wild type bases. The remainder of the base positions showed varying degrees of tolerance to base mutations with respect to their support of cis-cleavage. Evidence was obtained for the presence of a non-Watson-Crick base pair between A26 and G36 in the catalytic conformation of the hairpin ribozyme. On the basis of these results, a two-dimensional model for the hairpin ribozyme is presented.