Using the method of chromosomal walking, we have isolated a contiguous region of the Drosophila melanogaster X chromosome which corresponds to salivary gland chromosome bands 3C12 to 3D4. This five-band region contains approximately 100 kilobases of DNA, including those sequences comprising dunce, a gene which functions in memory and cyclic nucleotide metabolism. Genome blots of DNA from flies carrying several different chromosomal aberrations with breakpoints in the region have been probed with the isolated clones to map the breakpoints on the cloned DNA and to delimit dunce sequences. This has localized dunce to a 50-kilobase region. In addition, we have searched this 50-kilobase region for restriction site polymorphisms between X chromosomes from different Drosophila strains by genome blotting experiments, and we have followed the segregation of detected polymorphisms and dunce alleles after meiotic recombination. The data map one dunce mutation between two polymorphisms located 10 to 12 kilobases apart.