The positions of the nucleolus organizer regions in metaphase chromosomes of Drosophila hydei were detected by in situ hybridization experiments. In agreement with earlier conclusions the nucleolus of the X chromosome was found to originate in a terminal region of the heterochromatic arm. The Y chromosome contains two nucleolus organizers, one in a terminal postion of the long arm, and the other in the short arm. The implications with respect to the evolution of the Y chromosome are discussed.