The DNA sequence of the mitochondrial large subunit (LSU) rRNA gene of Schizosaccharomyces pombe has been determined. In the direction of transcription, this gene is located between the gene coding for subunit II of cytochrome oxidase and a cluster of three tRNA genes. Both the 5' and 3' ends of the LSU rRNA have been mapped precisely: whereas the 5' end can be assigned unambiguously to a single nucleotide position, multiple 3' ends occur within a run of eight U residues. Based on these results, the S. pombe LSU rRNA is between 2818 and 2826 nucleotides long. A sequence motif immediately upstream of the 5' end of the gene resembles that of the mitochondrial promoter motif of Saccharomyces cerevisiae; however, the sequence at the 3' end of the gene is not similar to any of the motifs implicated as processing signals in other mitochondrial systems. Unlike its counterparts in S. cerevisiae and Aspergillus nidulans, the mitochondrial LSU rRNA gene of S. pombe does not contain an intron. Comparison of potential secondary structure among the three fungal mitochondrial and Escherichia coli LSU rRNAs has defined a common secondary structure core, held together by long-range hydrogen-bonding interactions. A 5.8S-like structure is present within the 5'-terminal region of all three fungal mitochondrial LSU rRNAs; in contrast, no 4.5S-like structure is evident at the 3' end of these molecules. An evolutionary evaluation of highly conserved regions of a small set of LSU rRNA sequences suggests that S. pombe mitochondria diverged from a mitochondrial proto-fungal branch earlier than either A. nidulans or S. cerevisiae mitochondria. This result, considered in conjunction with the patterns of genome organization and codon usage in fungal mitochondria, points to a slower evolutionary clock speed in the mitochondrial genome of S. pombe.