In order to identify conserved potential secondary structures within translational start sites, mRNA sequences derived from different species were studied with programs able to depict such features. The potential secondary structure of 71 bases around the initiator AUG or AUGs in the coding sequences of 290 eukaryotic mRNAs was first examined and compared to 290 similarly analyzed regions derived from prokaryotic mRNA sequences (Nucleic Acids Res (1987) 15, 345-360). In both sets of sequences the initiator codon was often found to be in an open potential structure whereas a denser region characterized by nearly-periodic spacings defined the coding regions. Randomization of the sequences obliterated the observed patterns suggesting that the structure of the mRNA may determine these differences. Three sets of eukaryotic and prokaryotic mRNAs of approximately equal length were analyzed and found to preserve an open unpaired non-coding region 5' to the start codon. The start codon was found free of potential secondary structure in over 80% of all the sequences analyzed. These data, and study of mutants that restrict the accessibility of the start codon to the ribosomal initiation complex, suggest that both the prokaryotic and eukaryotic mRNA start sites must occur free of potential secondary structure for efficient initiation. A striking difference of the eukaryotic mRNA sequences analyzed was the high propensity of the coding region vicinal to the start codon to form secondary structures. Certain translation-defective mutants exhibit impaired formation of these secondary structures suggesting that the structure of the coding regions adjacent to the start codons of eukaryotic mRNAs may be an important, thus far unexamined, determinant of initiation. We propose that, for all genes studied, the transition in secondary structure between the coding and non-coding regions may be an important determinant of initiation.