Incubation of abbreviated tRNA's (tRNA-C-COH's) with (chemically) preaminoacylated P1, P2-di(adenosine 5'-)diphosphates in the presence of purified RNA ligase effected transfer of an aminoacyladenylate moiety to the 3'-terminus of the abbreviated tRNA's in good yield. Aminoacylated (or misacylated) tRNA's may thus be prepared from fractionated or unfractionated tRNA-C-COH's; each of the five aminoacylated dinucleoside diphosphates tested was utilized as a substrate by RNA ligase. That the resulting "chemically aminoacylated" tRNA's were identical with those prepared by enzymatic aminoacylation was judged by comparison of 1) chromatographic properties on benzolated diethylaminoethyl-cellulose, 2) rates of chemical deacylation, and 3) affinities for elongation factor Tu, as well as 4) the ability of misacylated tRNA's so derived to be deacylated chemically and then reactivated enzymatically with their cognate amino acids.