To determine whether sequence context influences the ability of an AUG triplet to be recognized as an initiator codon by eukaryotic ribosomes, single nucleotide changes were introduced near the translational start site in a cloned preproinsulin gene. Maximum synthesis of preproinsulin occurred when a purine, preferably adenosine, was located three nucleotides upstream from the initiator codon. Adenosine is found most frequently in that position in natural mRNAs.