When high molecular weight Bacillus subtilis DNA is denatured, renatured and then examined by electron microscopy the main renatured product seen is a long duplex, usually with a single strand at each end, due to in-register renaturation. In addition, structures containing short duplex segments of length 4830 ± 250 base pairs, with two single-strand arms at each end, are seen at a low frequency. Several lines of evidence support the hypothesis that these short duplex segments are formed by out-of-register renaturation of the 16 S + 23 S ribosomal RNA genes (rDNA) of B. subtilis. They are of the correct length. Their formation is inhibited if homologous, but not if heterologous, ribosomal RNA is added to the hybridization mixture. The frequency of occurrence of the duplex structures is consistent with the rDNA hypothesis. Heteroduplex molecules are seen with two or three rDNA duplex segments separated by single-strand substitution loops with specific lengths for each of the two single-strand arms of any one loop. On the basis of these structures, linkage groups containing seven or nine rDNA sets (each set containing one 16 S and one 23 S rDNA gene) separated by spacer DNA's are proposed. All of the 16 S rDNA genes are linked to 23 8 rDNA and vice versa with little or no spacer DNA between a 16 S and 23 S sequence. If 5 S DNA is present in the set, any spacer between it and the other ribosomal RNA gene must also be short. The prophage SPO2 bacterial att site maps at a distance of 6200 bases away from a 16 S + 23 S rDNA set, which is itself separated by a very short spacer (less than 600 bases) from a second rDNA set. © 1973.