In an approach to the functional anatomy of the mitochondrial genome and its evolution, we have compared buoyant densities, contour lengths, and denaturation maps in circular mitochondrial DNAs of the genus Drosophila. Mitochondrial DNAs from three representatives of the subgenus Drosophila (D. virilis, D. hydei, D. funebris) are similar in size (approx. 5 mum or 1 - 10(7) daltons) and buoyant density (approx. 1.685 g/ml), while in two members of the subgenus Sophophora (D. melanogaster, D. simulans), mitochondrial DNAs are longer (approx. 6 mum or 12.4 - 10(6) daltons) and have a lower buoyant density (approx. 1.681 g/ml). The latter mitochondrial DNAs also share one distinctly large early melting region, which in D. melanogaster is equivalent to 1.54 mum of native DNA. The corresponding (A + T)-rich region in D. virilis or D. hydei mitochondrial DNA is 1 mum shorter. Except for this region, denaturation maps of D. melanogaster and D. virilis mitochondrial DNAs are indistinguishable. The addition or deletion of a single block of (A + T)-rich sequences can fully account for the differences in buoyant density and size between the mitochondrial DNAs we have examined. In an appendix, we show that there is an equivalent discrepancy between the extent of strand separation determined by electron by electron microscopy and the actual extent of DNA denaturation, whether this is determined from absorbance changes or inferred from the reduction in contour lengths of individual circular molecules. The reduction in contour length appears to result exclusively from the uniform foreshortening of single-stranded DNA, not only in regions of visible strand separation but also in denatured regions hidden within putatively native segments of molecules. For molecules showing 15--45% strand separation, we estimate that putatively native segments are approximately 50% denatured.