Long pyrimidine tracts, purified from Drosophila melanogaster DNA after treatment with formic acid-diphenylamine, were used as template for E. coli RNA polymerase to produce a polynucleotide containing only purines. This polypurine RNA hybridized specifically to D. melanogaster DNA with high efficiency at low Cot values. The resulting hybrid showed high thermal stability. When polypurine RNA was subjected to complete hydrolysis with ribonuclease T1, over 90% of the nucleotide products were ApGp and ApApGp. Partial hydrolysis yielded a distinct additional component, ApApGpApGp + ApGpApApGp. We conclude that the major sequence in the polypurine transcript is (ApGpApApGp)n. In situ hybridization to salivary gland polytene chromosomes and to metaphase chromosomes from neural ganglia indicated that polypyrimidines complementary to polypurine RNA are located in heterochromatin. In femal cells, the predominant labeling was on centromeric heterochromatin of the 2nd chromosome. We have verified the location of polypyrimidines in neural ganglion cells, by using a cytological marker of chromosomes 2. In male cells, hybrid was also found on the Y chromosome.