The proximal half of the A (= X) chromosome of D. madeirensis has a gene arrangement very similar to the A1 or A6 inversions found in D. subobscura. Polytene chromosome analysis of hybrids between D. madeirensis and strains of D. subobscura homozygous for such inversions shows, however, that D. madeirensis has a gene arrangement different from any known for D. subobscura. These results provide evidence for a greater differentiation of the X chromosome in these species than has previously been described; it seems that the X chromosome is the only one that has undergone structural variation during the speciation process.