The influence of the nucleotide at position -3 relative to the AUG initiation codon on the initiation of protein synthesis was studied in two different in vitro translation systems using synthetic mRNAs. The four mRNAs, transcribed from cDNAs directed by an SP6 promoter, were identical except for mutations at nucleotide -3. In each case, translation of mRNAs produced a single protein of Mr = 12,600. Relative translational efficiencies showed a hierarchy in the reticulocyte lysate system (100, 85, 61 and 38% for A, G, U and C in position -3, respectively) but no differences in the wheat germ system. Differential mRNA degradation or polypeptide chain elongation were excluded as causes of the differences observed in translation in the reticulocyte lysate. mRNA competition increased the differences observed in translational efficiencies in reticulocyte lysate but showed no effect in wheat germ. Analysis of 61 plant and 209 animal mRNA sequences revealed qualitative and quantitative differences between the consensus sequences surrounding AUG initiation codons. Whereas the consensus sequence for animals was CACCAUG that for plants was AACAAUGGC. Both the structural and functional findings suggest that the factors which select AUG initiation codons in plants and animals differ significantly.