With the exception of a few genes, most of the mitochondrial (mt) genome of Pneumocystis carinii has not previously been sequenced. Shotgun sequences generated as a result of the Pneumocystis Genome Project (PGP) were assembled with the gap4 assembly program into a 23-kb contig. Annotation of the mt genome identified 4 open reading frames and 20 tRNAs in addition to 17 other genes: ATP synthase, subunits 6, 8, and 9; cytochrome c oxidase, subunits 1, 2, and 3; NADH dehydrogenase, subunits 1, 2, 3, 4, 4L, 5, and 6; apocytochrome b; RNase P RNA gene; and the mitochondrial large and small ribosomal RNA subunits. A 24-bp unit that repeated from one to five times was identified interior to the ends of the mt genome. Migration of the genome on CHEF gels was consistent with that of linear DNA and digestion with BAL31 showed a concomitant reduction in size of the genome, a characteristic of linear DNA. Together with the identification of terminal repeats similar to those found in other linear fungal mt genomes and the inability to join the ends by PCR, these data provide strong evidence that the mt genome of P. carinii is linear.