A new method is described for the detection of tRNA in complex with an aminoacyl tRNA synthetase. Escherichia coli isoleucyl tRNA synthetase complexes only with tRNA Ile and tyrosyl tRNA synthetase complexes exclusively with tRNA Tyr . ATP and amino acid are not required to establish the complex, which also forms equally well whether the tRNA is acylated or not. Quantitative analysis of the binding curves suggests a single binding site per enzyme particle which binds either aminoacylated or unacylated tRNA, both tRNA's having an association constant of approximately 10 8 liters/mole. The binding technique also provides a short and direct procedure for obtaining tRNA highly enriched for a selected acceptor.