Abstract In S. cerevisiae, ribosomal protein L32 regulates the splicing of the transcript of its own gene, RPL32. We have identified an RNA structure within the transcript that is responsible for this regulation. Initial deletions limited essential sequences to the 5′ exon and the first few nucleotides of the intron. To take advantage of phylogenetic comparison of RNA structures, RPL32 was cloned from the closely related species, Kluyveromyces lactis. The splicing of Its transcript is similarly regulated. Sequences conserved between the S. cerevisiae and K. lactis transcripts suggested a structure involving base pairing of a region encompassing the 5′ splice site with another nearthe 5′ end of the transcript. Analysis of numerous site-directed mutations supports this structure. We infer that stabilization of this structure by L32 inhibits splicing by precluding the interaction of U1 RNA with the 5′ splice site.