Abstract A method was developed for the chromatographic separation of soluble ribonucleic acid (sRNA) and ribosomal ribonucleic acid (rRNA). It involves the use of Sephadex G-75, which was equilibrated with 1.5 M NaCl and 0.5 M Tris buffer, pH 7.5. After the mixture was embedded, the column was eluted with 0.5 M Tris buffer using a 50 ml mixing pot. Excellent separation of sRNA and rRNA was obtained from purified samples as well as crude samples isolated from a human leukemic lymphoblast. Column recoveries were essentially 100%, and the method is fast and very reproducible. Other advantages, such as column capacities, non-denaturation and completeness of separation are discussed.