Cleavage of D. melanogaster rDNA with the Eco R1 restriction endonuclease reveals two major classes of repeating units: a long class of 17 kilobases (kb) and a short class of 11.4 kb. R loop mapping has been used to determine the topography of the sequences corresponding to the 18S and 28S rRNAs in both classes, including a cloned member (Dm103) of the long class. This mapping procedure derives from a novel reaction that we discovered between single-stranded RNA and homologous regions in duplex DNA molecules. In high formamide and at an elevated temperature, the RNA pairs with one of the two DNA strands in the region of homology to form an R loop in which one element is an RNA/DNA duplex and the other is single-stranded DNA (Figure 1). Mapping is accomplished by visualization of R loops in the electron microscope. The R loop map of Dm103 parallels that determined independently by Glover and Hogness (1977) from an analysis of its restriction fragments. Both maps indicate that the 28S rDNA in this cloned unit is divided into two blocks by a 5 kb insertion segment. R loop mapping of a population of long units obtained directly from the rDNA (that is, without cloning) has demonstrated that this interruption of the sequence coding for the 28S rRNA is a characteristic of the long class. By contrast, the 28S rDNA in the short units that we examined is not interrupted by an insertion segment. Otherwise, the R loop maps of the long and short units do not differ significantly. The two classes therefore correspond to repeating units that do (IN+) or do not (IN-) contain an insertion segment. Models for the transcription and function of these two classes of repeating units are discussed.