Partial deletion of the exon 5' to S. cerevisiae intron a5, a self-splicing mitochondrial class II intron, reveals the existence of several sites of intron-exon interaction. We have identified two of the corresponding exon-binding sites in intron a5 by comparative sequence analysis and RNAase H digestion of the intron complexed to a DNA version of its 5' exon. Introduction of mutations in either the intronic sites or the complementary exonic sequences affects splicing in vitro, whereas double mutants in which intron-exon pairings have been restored show normal activity. Some of the mutants accumulate a product that was shown to be the intron-3' exon lariat, a postulated splicing intermediate. The possible role of one of the intronic sites in aligning exons for the ligation step is discussed.