A series of Mu-1 induced isoleucine and valine auxotrophs derived from the wild type K-12 strain of Escherichia coli and from a valine resistant (ilvO-) mutant were examined. It was concluded that the genes ilvE, ilvD and ilvA constitute a single operon and are transcribed from E to A. Furthermore, the ilvG gene, expressed only in ilvO- strains, does not lie between ilvE and ilvD as previously assumed. A mutation in rho was examined for its effect on the ilvEDA operon. One effect of the rho- mutation was a mimicking of an isoleucine limitation signal. A model for the regulation of the ilvEDA operon is discussed. The model involves multiple attentuation sites and a possible role for the ilvO locus which lies at the distal end of the ilvEDA operon (but is not part of it). Supportive evidence for the proposed direction of transcription was obtained by examination of a series of gammailv transducing phages.