We had previously used RNAse A digestion to analyse (N-acetyl)-aminoacylation of RNA. 14 RNAse A cleaves the terminal adenosine nucleoside of the acceptor stem whilst maintaining any aminoacyl ester bonds. By comparison against synthetic standards, the 2'/3'-(N-acetyl)-Ala-ester adenosines could be identified in the digestion products of (N-acetyl)-aminoacylated RNA when analysed by HPLC, and thus the presence of terminal 2'/3'-(N-acetyl)-aminoacyl esters in the undigested RNA could be deduced.