Sequences flanking the AUG initiator codon influence its recognition by eukaryotic ribosomes. From a comparison of several hundred mRNA sequences, CC A G CCAUGG emerged as the consensus sequence for initiation in higher eukaryotes. Systematic mutagenesis of a cloned preproinsulin gene confirmed the facilitating effect of A or G in position− 3 (ie 3 nucleotides upstream from the AUG codon). C in positions− 1 and− 2, and G immediately following the AUG codon. The analysis of a new set of mutants now reveals that sequences slightly farther upstream are also influential, the optimal context for initiation being (GCC) GCC A G CCAUGG. Possible mechanistic implications of the repeating GCC motif are discussed.