Restriction-map variation in 64 X chromosome lines extracted from three different natural populations of Drosophila melanogaster was investigated with seven six-nucleotide-recognizing enzymes for a 20-kb region including the zeste and tko genes. Ten restriction-site and four length polymorphisms (two insertions and two deletions) were detected. Contrary to the predicted lower level of variation for genes on the X chromosome, the level of variation attributable to nucleotide substitution (estimated heterozygosity/nucleotide = 0.004) was similar to that previously reported for autosomal loci. The amount of insertion/deletion variation in the studied region was within the range observed in autosomal regions and thus not explainable by a simple selection model against the effects of insertional mutations. A general lack of linkage disequilibrium between polymorphic sites was observed.