Sixteen single point mutations near the beginning of the lacZ gene have been isolated and their effect on lacZ expression has been measured. Five mutations were obtained that alter a potential stem-and-loop structure in the messenger RNA that masks the initiation codons. Formation of this stem-and-loop is a result of transcription of DNA sequences introduced during the cloning of the lac regulatory region. The mutations isolated were then moved into a background that deleted this structure. Analysis of these mutations indicated that the secondary structure inhibited lacZ expression 5.8-fold and that either single point mutations or a 9 base-pair deletion could relieve this inhibition completely. In addition, it was found that an A to C transversion in the first base following the initiation codon (in the absence of the inhibitory secondary structure) decreases lacZ expression almost twofold, whereas C to U transitions in the next two positions have negligible effects. Mutations were also obtained that either increase or decrease the length of the Shine-Dalgarno sequence. The effects of these mutations were studied in the presence or absence of the secondary structure that involves the two initiation codons. It was found that when translation initiation was inhibited by the secondary structure, increasing the length of the Shine-Dalgarno sequence increased lacZ expression 2.8-fold and decreasing the length of this sequence reduced lacZ expression 12-fold. When translation initiation was not inhibited by the secondary structure, increasing the length of the Shine-Dalgarno sequence had no effect and decreasing the length of this sequence only reduced lacZ expression sixfold. The mechanistic implications of these results are discussed. Two initiation codons are located in the beginning of the lacZ gene, 7 and 13 bases from the Shine-Dalgarno sequence. NH2-terminal sequence analysis indicated that the majority of the protein synthesized initiate at the first initiation codon in the wild-type lacZ gene (in agreement with results reported previously by J. L. Brown and his colleagues). Upon introduction of sequences that result in a change in the mRNA secondary structure, both initiation codons are used in almost equal amounts. Three mutations and two pseudorevertants were obtained, which are located in the first initiation codon. It was found that when the first initiation codon is changed from AUG to GUG, translation initiation is decreased tenfold at that codon. Other mutations at the first initiation codon resulted in initiation occurring exclusively at the second initiation codon at an efficiency that is determined by the nucleotides located in the spacer region.