We have studied the consequences of mutational alteration in the structure of EF-Tu on the missense errors and proofreading activity of bacterial ribosomes in vitro. Our data show that the EF-Tu Bo mutant form of EF-Tu (van der Meide et al. 1983a) is inactive in polypeptide synthesis on the ribosome, even though it binds aminoacyl-tRNA. A second mutant form, EF-Tu Ar (van der Meide et al. 1983a), is active in polypeptide synthesis but supports a much higher messense incorporation with either leucine isoacceptor 2 or leucine isoacceptor 4 in the in vitro system. Further analysis of the kinetic basis of this enhanced missense frequency revealed that the mutation responsible for the alteration in EF-Tu Ar increases the errors at both the proofreading step and the initial selection. In this respect the effect of this particular mutation is similar to the mode of action of the antibiotic kanamycin (Jelenc and Kurland 1984).