The empoyment of a novel method of affinity chromatography, which makes use of antibodies that specifically bind DNA/RNA hybrids, has made it possible to enrich for rat rDNA molecules which contain R loops formed with the 18S and 28S rRNAs. An approximately 150-fold enrichment of the ratrRNA coding sequences was obtained by this affinity chromatography procedure. This degree of enrichment made it possible to visualize these R loop containing molecules in the electron microscope and, thus, to obtain a map of the transcribed and spacer regions of rat rDNA. Eleven of the molecules that were observed contained either 3 or 4 R loops, or else 2 R loops separated by a long spacer. Thus, these molecules provided direct information in regard to the length of rat rDNA repeating units. The mean length of the repeating units was 37.2 kbp with a standard deviation of 1.3 kbp. Within the errors of the measurements, these could all represent repeating units of exactly the same length, although a certain degree of length heterogeneity, possibly up to 4 or 5 kbp, cannot be ruled out by the data. If significantly longer or shorter rDNA repeating units exist in the rat genome, they are probably much less common than the 37.2 kbp unit. These electron microscopic measurements provide the most definitive data yet available on the size of the repeating units of mammalian rRNA genes.