Seventeen biotin-labeled DNA sequences were hybridized to polytene chromosomes of Drosophila melanogaster and D. funebris in order to establish chromosomal homologies between these species. Ten probes correspond to cloned DNA sequences from D. melanogaster (RpII 215, MHC, H3-H4, Tor, hsp 68, hsp 28/23, hsp 83, PP1alpha, RpII 140, and ey), four are clones isolated from a D. subobscura genomic library (Xdh, lambdaDsubS3, lambdaDsubG3, and lambdaDsubG4), two are clones from D. funebris (F2 and Adh) and one from D. virilis (ci). The probes were chosen in order to cover all the autosomes, since X-chromosome homologies have already been studied by linkage analysis of morphological mutants. Most probes gave a unique hybridization signal; consequently, our results allow unambiguous inferences about chromosomal homologies. The results show extensive gene rearrangement within all chromosomal elements, probably due to paracentric inversions, but are consistent with Muller's proposal that chromosomal elements have conserved their genetic content during the evolution of Drosophila.