The location of the 5S ribosomal RNA cistrons in band 2-23B1,2 of the polytene (salivary gland) chromosomes of Drosophila hydei was indicated by in situ hybridization of tritiated low molecular weight RNA fractionated from total in vivo synthesized larval RNA or from in vitro synthesized salivary gland RNA and competition of the hybridization of this RNA by 5S RNA obtained from calf lens ribosomes. -- At the submicroscopic level, band 2-23B1,2 in salivary gland chromosomes shows a compact organization. The adjacent region, 23B2, is slightly puffed and displays typical RNP particles, some of which may be observed close to band 2-23B1,2.