The sequence arrangement of ribosomal DNA (rDNA) spacers in Drosophila melanogaster was analyzed with restriction endonucleases. Spacers, derived from cloned rDNA repeats and from uncloned purified rDNA, are internally repetitive, as demonstrated by the regular 250 base pairs interval between sites recognized by the enzyme Alu I. Length heterogeneity of spacers is due at least in part to varying numbers of repeated sequence elements. All spacers and analyzed, whether derived from X or from Y chromosomal rDNA, have a very similar sequence organization. The distance separating the repeated nontranscribed spacer sequences from the 5' end of the transcribed region is conserved in all ten cloned fragments examined, and is probably less than 150 base pairs, as measured by electron microscopy.