Plasmids have been constructed containing reiterated copies of a 66 bp fragment, loosely referred to as the ribosome binding site, that includes the AUG initiator codon of preproinsulin. The extreme test involved plasmid 255/17, which carried four tandem copies of the ribosome binding site, with all four AUG triplets in the same reading frame as the preproinsulin coding sequence downstream. Initiation at any potential start site would generate a polypeptide precipitable with anti-insulin antiserum, and its size would reveal the AUG(s) active in initiation. One insulin-related polypeptide was synthesized in cells transfected by p255/17; its size corresponded to the product initiated at the first ribosome binding site in the tandem array. Inasmuch as the three downstream AUG triplets, which are not used, occur in a sequence context identical with that around the 5'-proximal AUG triplet, which is used, the position of an AUG triplet relative to the 5' end of the mRNA appears to be important in identifying it as a functional initiator codon.