A method for mapping all base-paired stems in both elongation and initiator tRNAs is described using double-stranded-specific ribonuclease V1 from the venom of the cobra Naja naja oxiana. 32p-end-labeled RNA is first partially digested with double-strand-specific V1 nuclease under near physiological conditions, and the resultant fragments are than electrophoretically fractionated by size in adjacent lanes of a polyacrylamide gel run in 90% formamide. After autoradiography, the base-paired nucleotides are definitively located by comparing V1 generated bands with fragments of known length produced by both Neurospora endonuclease and base-specific ribonucleases. Using the substrates yeast tRNAPhe an E, coli tRNAfMet of known three-dimensional structure, we find V1 nuclease to cleave entirely within every base-paired stem. Our studies also reveal that nuclease V1 will digest paired nucleotides not hydrogen-bonded by standard Watson-Crick base-pairing. In yeast tRNAPhe cleavage of both wobble base-pairs and nucleotides involved in tertiary base-base hydrogen bonding is demonstrated.