The toromere previously found by other workers in the distal end of the sixth chromosome (microchromosome) ofD. limmeiwas studied using differential staining ofDrosophila lummeigiant chromosomes. The toromere which was first described as a quinacrine-bright structure appears as a C-positive body. Quantitative cytofluorometric analysis showed highly significant increase in toromeric DNA under low temperature conditions.In situhybridization of125I nick-translatedD. virilissDNA (all three satellites were included in the sample) with polytene chromosomes ofD. lummeilarvae cultured at 12° C revealed no label incorporation into toromere region. Howeverin situhybridization of3H-RNA complementary to highly repetitious DNA ofD. lummeiandD. virilis(Cot = 10−1–10−2) with polytene chromosomes of the larvae cultured at 12° C coupled with banding studies enable us to conclude that the toromere probably contains an AT-rich repeated DNA. A well-developed toromere in the sixth chromosome of D.lummeiwas also demonstrated at normal temperature (25° C) in interspecific hybrids. The role of the toromere structure in the mitotic behavior of microchromosomes and their replication pattern is discussed.