Cs, a gene with unknown function, and amd and Ddc, which encode decarboxylases, are among the most closely spaced genes in D. melanogaster. Untranslated 3' ends of the convergently transcribed genes Cs and Ddc are known to overlap by 88bp. A number of questions arise about the organization of this tightly-packed gene region and about the evolution and function of the Cs gene. We have now investigated this three-gene cluster in Scaptodrosophila lebanonensis (which diverged from D. melanogaster 60-65 MYA), as well as in D. melanogaster and D. simulans. Gene order and direction of transcription is the same in all three species. The Cs gene codes, in Scaptodrosophila, for a polypeptide of 544 amino acids; in D. melanogaster, it consists of 504 amino acids, which is twice as long as previously suggested, which makes the gene density even more spectacular. The Cs sequences exhibit higher number of non-synonymous substitutions between species, higher ratios of non-synonymous to synonymous substitutions, and lower codon usage bias than other genes, suggesting that Cs is less functionally constrained than the other genes. This is consistent with the failure of inducing phenotypic mutations in D. melanogaster. The function of Cs remains to be identified, but a high degree of similarity indicates that it is homologous to genes coding for a corticosteroid-binding protein in yeast and a polyamine oxidase in maize.