The mitochondrial genome of Drosophila yakuba is a circular DNA molecule of 16019 nucleotide pairs. The sequence contains the genes for two rRNA molecules, 22 tRNA molecules, five known polypeptides (cytochrome b, cytochrome c oxidase subunits I, II, III and ATPase subunit 6) and eight unidentified polypeptides (URF1, 2, 3, 4L, 4, 5, 6 and A6L). Between the tRNA(ile) and small rRNA genes there occurs a sequence of 1077 nucleotides that is 92.8 per cent A + T and lacks reading frames greater than 123 nucleotides. Replication of the molecule originates in this A + T-rich region and proceeds toward the small rRNA gene. Non-coding nucleotides between genes are either absent or occur in low numbers (1 to 31). A sequence equivalent in size and secondary structure potential to the sequence associated with the initiation of second strand synthesis in mammalian mtDNA is missing in Drosophila mtDNA. While the genes found in D. yakuba and mammalian mtDNAs are the same, the relative arrangement of many of these genes differs considerably in the two molecules. The proportions of the two strands of the D. yakuba molecule which serve as template for transcription of genes are approximately equal. This contrasts with the situation in mammalian mtDNAs where all genes except those for URF6 and eight tRNAs are transcribed from one strand. The dihydrouridine and T psi C loops of D. yakuba mt-tRNA genes are highly variable in size, and among these genes there is a general deficiency of nucleotides which are highly conserved in prokaryotic and eukaryotic nuclear-coded tRNAs. The D. yakuba tRNA(AGYser) gene is unusual in that an eleven nucleotide loop replaces the dihydrouridine arm. D. yakuba mitochondrial polypeptide genes utilize 59 sense codons. However, 93.8 per cent of all codons used end in A or T. Unique variations occur in the Drosophila mitochondrial genetic code. AGA appears to specify serine rather than arginine as in the standard code, or termination as in the mammalian mitochondrial code. The Drosophila COI gene lacks a standard translation initiation codon, and may utilize a four nucleotide codon ATAA for that purpose. As in other metazoan mitochondria, TGA and ATA specify tryptophan and methionine, respectively. As a tRNA with an anticodon (TCT) specific for AGA codons does not appear to be encoded in D. yakuba mtDNA, it seems likely that the GCU anticodon of the D. yakuba tRNA which recognizes AGY (serine) codons can also recognize AGA.(ABSTRACT TRUNCATED AT 400 WORDS)