We have characterized the mitochondrial transcription factor (Mtf1) and RNA polymerase (Rpo41) of Schizosaccharomyces pombe. Deletion mutants show Mtf1 or Rpo41 to be essential for cell growth, cell morphology and mitochondrial membrane potential. Overexpression of Mtf1 and Rpo41 can induce mitochondrial transcription. Mtf1 and Rpo41 can bind and transcribe mitochondrial promoters in vitro and the initiating nucleotides were the same in vivo and in vitro. Mtf1 is required for efficient transcription. We discuss the functional differences between Mtf1 and Rpo41 of S. pombe with Saccharomyces cerevisiae and higher organisms. In contrast to S. cerevisiae, the established model for mitochondrial transcription, S. pombe, a petite-negative yeast, resembles higher organisms that cannot tolerate the loss of mitochondrial function. The S. pombe and human mitochondrial genomes are similar in size and much smaller than that of S. cerevisiae. This is an important first step in the development of S. pombe as an alternative and complementary model system for molecular genetic and biochemical studies of mitochondrial transcription and mitochondrial-nuclear interactions. This is the first systematic study of the cellular function and biochemistry of Rpo41 and Mtf1 in S. pombe.