The transcription unit containing the yeast mitochondrial 21 S rRNA gene was analyzed by hybridizing purified 21 S rRNA and its precursors to restriction enzyme digests of mitochondrial DNA. The 21 S rRNA gene and several precursors were localized in a detailed restriction enzyme map of the 12-kb mitochondrial genome of petite F11. This petite contains a region of the mitochondrial genome encompassing the 21 S rRNA gene and five tRNA genes. The full transcription unit includes two 21 S exons (totaling 3.7 kb), an 0.8-kb intron, and a 1.0-kb extension on the 3′-end. The 3′-extension extends through the tRNA thr gene and probably also includes the tRNA his and tRNA cys genes. This transcription unit is surrounded by large nontranscribed regions; two additional F11 genes, for tRNA gln and tRNA leu, are not part of this transcription unit. Hybridization of total mitochondrial RNA indicates no difference between grande and petite yeast in the overall pattern of mitochondrial transcription from this region of the genome.