Chromosomal locations of the seven rRNA operons in Escherichia coli K-12 were studied by digesting DNA from various merodiploid strains with SalI restriction enzyme followed by Southern gel analysis with 32P-labeled 23S rRNA as a probe. The seven unique SalI DNA fragments revealed in the autoradiograms were first correlated to the seven rRNA operons previously isolated as hybrid plasmids or transducing phages. The chromosomal locations of six (rrnA, B, C, D, E, and G) of the seven isolated operons were confirmed by increased gene dosage demonstrated in autoradiograms after Southern gel analysis of DNA from relevant merodiploid strains. The gene dosage analysis showed that the location of the remaining operon (now called rrnH) is between metD and proA. No evidence was obtained for the presence of rrnF, which was previously reported to map between aroB and malA. The chromosomal location of rrnH was confirmed by P1 transduction in the following way: a DNA fragment adjacent to rrnH was cloned into pBR322; the resulting hybrid plasmid was integrated at the homologous region of the chromosome of a polA mutant; and the ampicillin resistance marker originally carried by pBR322 was then used for mapping of the nearby rrnH by P1 transduction. A close linkage of rrnH to metD (about 60% cotransduction) was observed, and the data were consistent with the order metD-rrnH-proA. Thus, mapping of all seven rRNA operons has been completed. The present study has also determined the orientation of rrnG and rrnH and demonstrated that the direction of transcription of all the rRNA operons is identical to that of DNA replication.