The genes for tranfer ribonucleic acid (tDNA) and 5S ribonucleic acid (5SDNA) were isolated from the total deoxyribonucleic acid (DNA) of Escherichia coli. The relatedness of tDNA and 5S from E. coli and other species of Enterobacteriaceae was determined by reassociation of the isolated genes labeled with 32PO4 to unlabeled, unfractionated DNA. Double-stranded DNA was separated from unreacted DNA by hydroxyapatite chromatography. Thermal elution profiles were done to determine the amount of unpaired bases present in related DNA sequences. Relative to total DNA, both 5S DNA and tDNA were highly conserved throughout the Enterobacteriaceae, including the genera Yersinia and Proteus.