A minor class of introns with noncanonical splice sites has been identified in both vertebrate and invertebrate genomes. The divergent consensus sequences within these introns suggest that splicing might be via a mechanism distinct from that used by the major class of introns. The low abundance U12 snRNA has been proposed to base pair with the predicted branch site sequence of these minor class introns, probably bulging out an adenosine to act as the nucleophile in the first step of splicing. We have identified homologues of the previously characterized human U12 snRNA in both mouse and chicken, where the minor class of introns has also been found. The U12 sequences that potentially base pair with the putative branch site are invariant. Additional conserved sequences at the 5' end of U12 snRNA could dynamically base pair with U6 snRNA sequences flanking the hexanucleotide ACAGAG to form structures analogous to those of three U2-U6 interactions genetically defined as important in the major class of spliceosome. We have also isolated two human U12 snRNA genes. One gene is functional for transcription of U12 snRNA, whereas the other appears to be a pseudogene. Sequences of the 3' box in both U12 snRNA genes are strikingly similar and bear high resemblance to those of U1 and U2 genes. Upstream elements, including the PSE and the DSE, have been identified and characterized in the functional gene. These features indicate that transcription of U12 snRNA is driven by RNA polymerase II.