IN 1964 Rosset, Monier and Julien1 described and characterized a low molecular weight ribonucleic acid (5S RNA) that is present in the ribosomes of Escherichia coli in addition to the two larger components, the 16S and 23S RNA. It contains 120 nucleotide residues and in contrast to transfer RNA contains no “minor” bases. Using a two-dimensional fractionation procedure for the separation of nucleotides labelled with phosphorus-32 (ref. 2), we have determined the sequences of all the oligonucleotides obtained by complete digestion of 32P-labelled 5S RNA with ribonuclease T1 and ribonuclease A (pancreatic ribonuclease)3. In order to arrange these nucleotides in the unique sequence of the 5S RNA, we have now studied the products of various partial ribonuclease digestions. This has involved the development of a number of new techniques which will be described in detail later. From the large number of partial digestion products obtained, we deduced the unique sequence shown in Figs. 1 and 2.