Cobra venom nuclease V1, which cleaves double-stranded RNA, has been used to study the structure of four Escherichia coli tRNAs: Phe, Glu2, Leu2, and Ile1. The cleavage patterns are compared to those found for yeast tRNAPhe, the three-dimensional structure of which is known. The cleavage patterns of all the tRNA molecules are similar, and the most sensitive cleavage is found at the central base pair of the anticodon stem. Studies of E. coli tRNALeu2, which has a large variable loop, are consistent with the formation of a base-paired stem and loop structure that is not closely bound to the remainder of the molecule. A survey of the results suggests that the V1 molecule may interact with the minor groove of the double helix with an affinity for stacked bases and that it may require two or three stacked bases for optimal binding and cleavage.