Rapid RNA sequencing technology was used to determine if the base specificities of an RNase recently purified from chicken liver would prove useful for RNA sequence analysis. Escherichia coli 5 S [5'-32P]rRNA or yeast 5.8 S [5'-32P]rRNA was digested with the enzyme and this digest, along with digests derived from RNases of known specificity (U2, T1, T2) were subjected to electrophoresis through denaturing polyacrylamide slab gels. Following autoradiography, the banding patterns arising from the activity of each enzyme were compared, and the base specificity of the unknown RNase was established. The chicken liver RNase was found to have a marked preference for phosphodiester bonds containing cytidylic acid residues, a property which should make the enzyme useful for distinguishing between pyrimidines in RNA sequencing.