We have mapped the single block of non-homologous sequences and measured the extent and distribution of base-pair substitutions within the homologous sequences in Drosophila melanogaster: Drosophila virilis heteroduplex mitochondrial DNAs (mtDNAs). Of the 4.8 kilobases long, unusually (A + T)-rich region in D. melanogaster mtDNA, only 0.5 kilobases can react with related, but not identical sequences in D. virilis mtDNA, while the rest (4.3 kilobases in the long arm of a heteroduplex loop) is replaced by a shorter, non-homologous region (1.0 kilobases in the short arm of the loop). No additional heterologous regions are evident. Homologous sequences have accumulated on the average 15.5% base-pair changes. Regionally, these substitutions are relatively uniformly distributed (14.5--16.5%) except for a single, more conserved region (10--13%), which presumably represents the ribosomal cistrons. The lack of general sequence stability suggests that the invariant topographic organization of the nucleotide sequence, previously recognized among Drosophila mtDNAs, is under more stringent selection than the sequence per se.