The sequence of a segment of the Drosophila virilis mitochondrial DNA (mtDNA) molecule that contains the A + T-rich region, the small rRNA gene, the tRNA(f-met), tRNA(gln), and tRNA(ile) genes, and portions of the ND2 and tRNA(val) genes is presented and compared with the corresponding segment of the D. yakuba mtDNA molecule. The A + T-rich regions of D. virilis and D. yakuba contain two correspondingly located sequences of 49 and 276/274 nucleotides that appear to have been conserved during evolution. In each species the replication origin of the mtDNA molecule is calculated to lie within a region that overlaps the larger conserved sequence, and within this overlap is found a potential hairpin structure. Substitutions between the larger conserved sequences of the A + T-rich regions, the small mt-rRNA genes, and the ND2 genes are biased in favor of transversions, 71-97% of which are A----T changes. There is a 13.8 times higher frequency of nucleotide differences between the 5' halves than between the 3' halves of the D. virilis and D. yakuba small mt-rRNA genes. Considerations of the effects of observed substitutions and deletion/insertions on possible nucleotide pairing within the small mt-rRNA genes of D. virilis and D. yakuba strongly support the secondary structure model for the Drosophila small mt-rRNA that we previously proposed.