This paper describes a method for the identification of single copy genes in Drosophila melanogaster polytene chromosomes, using fluorescence in situ hybridization (FISH). We demonstrate the detection of white (w), a gene previously mapped to 1-1.5 region of the linkage map, and to 3C2 region of the cytogenetic map of X chromosome. Squash preparations of polytene chromosomes from salivary glands dissected out from third instar larvae of Drosophila melanogaster were denatured and subjected to hybridization with a digoxigenin labeled probe, corresponding to mini-white gene. The preparations were then washed and incubated with anti-digoxigenin-fluorescein antibodies. After removal of the nonspecifically bound antibodies, the polytene chromosomes were counterstained with propidium iodide. Fluorescence microscopy revealed white locus in the X chromosome in a subterminal location, in agreement with the above mentioned maps. The protocol is efficient and adaptable for simultaneously multiple signal detection.