A group I intron from a bacterial tRNA precursor has been converted into an RNA enzyme that catalyzes the efficient polymerization of oligoribonucleotide analogs of tRNA exons using a reaction scheme consisting of multiple cycles of reverse and forward exon ligation reactions. Here, we present results showing that this system represents a novel and useful tool for the analysis of 3' splice site reactions of group I ribozymes. First, analysis of variant substrates containing base substitutions in group I secondary structure elements P1, P9.0 and P10 confirms that exon polymerization is dependent on these structures, and therefore constitutes an appropriate and relevant model system for studying the exon ligation step of splicing. Second, to probe interactions between the intron's catalytic core and the bases and backbone of the P1/P10 reaction helix, two successful strategies for separating the internal guide sequence from the intron core were devised. One such strategy uses a construct in which the reaction helix interacts functionally with the catalytic core using only tertiary contacts. Further stabilization of this interaction through the inclusion of a 7 bp intermolecular P2 helix generates increased reaction efficiency. Third, when provided with two reaction helices, the ribozyme synthesizes mixed polymers through a mechanism that involves sequential binding and release of the duplexes. Fourth, in these reactions, turnover of the external guide sequence requires unwinding and annealing of the P2 helix, suggesting that P2 unwinding may occur during group I splicing. These results provide novel experimental tools to probe the relatively poorly understood 3' splice site reactions of group I introns, and may be relevant to ribozyme-catalyzed assembly and recombination of oligomers in prebiotic scenarios.