N-Benzyl-D-amphetamine is a potent in vitro and in vivo inhibitor of phenylalanyl-tRNA synthetase of Escherichia coli. The concentration of this inhibitor necessary for the in vivo inhibition is approximately 100-fold greater than that necessary for inhibition of the purified enzyme. Treatment of rel+ strains of E. coli with the inhibitor results in a decreased percentage of tRNA Phe which is charged, guanosine tetraphosphate formation, cessation of RNA synthesis, and growth arrest. Evidence is presented which demonstrates that the primary and perhaps sole mode of action of N-benzyl-D-amphetamine is inhibition of phenylalanyl-tRNA synthetase.