The gene for the U6 small nuclear RNA (snRNA) in the fission yeast Schizosaccharomyces pombe is interrupted by an intron whose structure is similar to those found in messenger RNA precursors (pre-mRNAs) (1). This is the only known example of a split snRNA gene from any organism--animal, plant, or yeast. To address the uniqueness of the S. pombe U6 gene, we have investigated the structures of the U6 genes from five Schizosaccharomyces strains and three other fungi. A fragment of the U6 coding sequence was amplified from the genomic DNA of each strain by the polymerase chain reaction (PCR). The sizes of the PCR products indicated that all of the fission yeast strains possess intron-containing U6 genes; whereas, the U6 genes from the other fungi appeared to be uninterrupted. The sequences of the Schizosaccharomyces U6 gene fragments revealed that each had an intron of approximately 50 base pairs in precisely the same position. In addition to the splice sites and putative branch point regions, a sequence immediately upstream of the branch point consensus was found to be conserved in all of the Schizosaccharomyces U6 genes. This sequence matches the consensus for the B box of eukaryotic tRNA promoters. These results raise the interesting possibility that synthesis of U6 RNA in fission yeast might involve the use of internal promoter elements similar to those found in other genes transcribed by RNA polymerase III.