Abstract The mischarged species Val-tRNAile and Phe-tRNAile (both Escherichia coli B) were enzymatically synthesized in a heterologous aminoacylation system under special reaction conditions. When mixed with isoleucyl-tRNA synthetase in the absence of AMP or PPi, Val-tRNAile is very rapidly deacylated at pH 7, 5 mm Mg2+, 15°. On the other hand, isoleucyl-tRNA synthetase does not deacylate Phe-tRNAile. Isoleucyl-tRNA synthetase can form valyl adenylate (Val-AMP) from valine and ATP, but the isoleucyl-tRNA synthetase-bound Val-AMP is rapidly hydrolyzed when confronted with tRNAile (Baldwin, A. N., and Berg, P. (1966) J. Biol. Chem. 241, 839). Since Val-tRNAile is rapidly and specifically deacylated by isoleucyl-tRNA synthetase, it is reasonable to speculate that Val-tRNAile is a transient intermediate in the tRNAile-induced hydrolysis of Val-AMP. This possibility was tested further by kinetic studies of the Val-tRNAile deacylation and the tRNAile-induced hydrolysis of Val-AMP.