A 4-kilobase DNA fragment carried by a recombinant lambda gt11 bacteriophage appears to contain most of the coding information for the 145-kDa subunit of the Saccharomyces cerevisiae mitochondrial RNA polymerase. The RPO41 gene is located on chromosome VI, as determined by hybridization to electrophoretically separated yeast chromosomes. Hybridization and gene disruption/replacement experiments show that the RPO41 gene exists in a single copy and that its product is required for transcription and maintenance of the mitochondrial genome.