A conserved sequence element in a minor class of eukaryotic pre-messenger RNA (pre-mRNA) introns was previously proposed to base pair with a complementary sequence in the U12 small nuclear RNA (snRNA) in a manner analogous to the pairing of U2 snRNA with the branch site sequence of the major class of introns. Here, mutations generated in this conserved sequence element block the splicing of a member of this minor intron class in vivo. This block was relieved by coexpression of a U12 snRNA containing compensatory mutations that restore the proposed base pairing interaction, These results show that this minor class of pre-mRNA introns is a distinct class existing alongside the major class of introns in animal genomes, and these results also establish an in vivo function for U12 snRNA.