The genes coding for the large rRNAs (rDNA) were purified from Drosophila melanogaster embryos by two different methods. Purified rDNA was analyzed by gel electrophoresis after digestion with restriction endonucleases and by electron microscopy. Each repeating unit of rDNA consists of a gene region that codes for a transcript which includes the 18S and 28S rRNA sequences plus a nontrascribed spacer. Analysis of rRNA/rDNA hybrids in the electron microscope reveals that two types of repeating units exist in the rDNA from this fly. One of these is characterized by insertions of various lengths within the 28S gene. Insertions range in size from about 0.5 kilo base pairs (kb) to 6.0 kb and occur in distinct size classes which are multiples of 0.5 kb. Figure 10 summarizes the structure of the rDNA repeating unit. Repeats with insertions constitute about two thirds, and repeats without insertions about one third of all repeating units in the sample studied where 86% of the rDNA is derived from the X chromosome nucleolus organizer. Statistical analysis of pairs of of nearest-neighbor repeats demonstrates that repeats with and without insertions are interspersed in the X chromosome nucleolus organizer, and that scrambling is close to that expected for random arrangement. Insertions are the major source of length heterogeneity in this rDNA. A minor length heterogeneity also exists in thie nontranscribed spacer of Drosophila rDNA as demonstrated by heteroduplex mapping of restriction fragments in the electron microscope.