Enzymatic labeling of the 3′-linked terminus of MS2 ribonucleic acid (RNA) with polynucleotide kinase is possible only after phosphomonoesterase treatment of the nucleic acid. Pancreatic ribonuclease degradation of the labeled product results in the formation only of labeled trinucleoside tetraphosphate, indicating an initial sequence of two purines followed by a pyrimidine. Alkaline degradation of RNA bearing a uniform 32P label shows that in the virus the terminal nucleoside is present as the 5′-triphosphate. The natural initial trinucleoside hexaphosphate has been isolated from pancreatic ribonuclease digests and the structure was determined to be pppGpGpUp. © 1968, American Chemical Society. All rights reserved.