5-Azacytidine (5-AZ) profoundly affects gene expression and chromosome structure in higher eukaryotes, presumably by disrupting normal patterns of DNA methylation. The DNA of several eukaryotic species, including Drosophila melanogaster, is virtually devoid of 5-methylcytosine, and the spectrum of mutagenic effects induced by 5-AZ in such organisms is less well characterized. To investigate the mutagenicity of 5-AZ in Drosophila germ cells, DNA repair-deficient (mei-9) Drosophila 72-hr-old larvae were fed on medium containing 5-AZ, and recovered adult males were tested for induced losses and interchanges involving the paternal sex chromosomes. Moderately toxic doses of 5-AZ were found to induce significant rates of apparent complete losses (CL) of the paternal sex chromosomes, partial losses (PL) of one of the arms of the submetacentric Y-chromosome, and interchanges (X-Y) between the X-chromosome and the short arm of the Y-chromosome. The data suggest that, of the stages tested, germ cells in the early primary spermatocyte stage of development are maximally sensitive to 5-AZ-induced sex chromosome loss and rearrangement. X-Y interchanges comprised a substantial fraction of the 5-AZ-induced breakage events involving the Y-chromosome; in contrast with classical clastogens such as X-rays, the pattern of interchange products suggests that 5-AZ acts by enhancing the frequency of pairing-dependent interchanges between the paternal sex chromosomes.