The authors describe a method to determine the positions of adenines, guanines, and pyrimidines in RNA sequences by partial nuclease digestion and electrophoretic fractionation. Terminal labeling with 32P is used to establish a reference point at one end of the RNA molecule. RNase T1 cleaves at guanines, while RNase U2 cleaves only at adenines. Limited alkaline hydrolysis provides a continuum of fragments from breaks at every phosphodiester bond. These reactions are performed on yeast 5.8S ribosomal RNA, and the products are fractionated by size on a polyacrylamide gel. Autoradiography of the gel displays the sequence up to 100 nucleotides from the end of the molecule, though uracil cannot be distinguished from cytosine. The technique forms the basis of an RNA sequencing method. The authors also discuss the limitations and future directions of the method, noting that pancreatic RNase does not yet distinguish uracil from cytosine.The authors describe a method to determine the positions of adenines, guanines, and pyrimidines in RNA sequences by partial nuclease digestion and electrophoretic fractionation. Terminal labeling with 32P is used to establish a reference point at one end of the RNA molecule. RNase T1 cleaves at guanines, while RNase U2 cleaves only at adenines. Limited alkaline hydrolysis provides a continuum of fragments from breaks at every phosphodiester bond. These reactions are performed on yeast 5.8S ribosomal RNA, and the products are fractionated by size on a polyacrylamide gel. Autoradiography of the gel displays the sequence up to 100 nucleotides from the end of the molecule, though uracil cannot be distinguished from cytosine. The technique forms the basis of an RNA sequencing method. The authors also discuss the limitations and future directions of the method, noting that pancreatic RNase does not yet distinguish uracil from cytosine.