Twelve biotin-labelled recombinant DNA probes were hybridized to polytene chromosomes of Drosophila melanogaster and D. lebanonensis. Probes were chosen in order to cover the whole chromosomal complement. Six probes correspond to known genes from D. melanogaster (RpII215, H3-H4, MHC, hsp28/23, hsp83, hsp70), four probes are clones isolated from a D. subobscura library (Xdh, lambda DsubS3, lambdaDsubG3, lambdaDsubG4) and the remaining two probes correspond to the Adh gene of D. lebanonensis and to one sequence (262), not yet characterized, from the same species. The chromosomal homologies obtained from the in situ hybridization results allow us to determine that Muller's C and D chromosomal elements are fused in the karyotype of D. lebanonensis and constitute the large metacentric chromosome. Single pericentric inversions in the E and B elements have generated the medium and small metacentric chromosomes, respectively. No great changes are detected in Muller's A element, which remains acrocentric. The changes detected in the karyotypic evolution of D. lebanonensis are frequently observed in Drosophila evolution, as deduced from chromosomal homologies of several Drosophila species. The results are also consistent with Muller's proposal that chromosomal elements have been conserved during the evolution of Drosophila.