In a lacZ expression vector (pMC1403Plac), all 64 codons were introduced immediately 3' from the AUG initiation codon. The expression of the second codon variants was measured by immunoprecipitation of the plasmid-coded fusion proteins. A 15-fold difference in expression was found among the codon variants. No distinct correlation could be made with the level of tRNA corresponding to the codons and large differences were observed between synonymous codons that use the same tRNA. Therefore the effect of the second codon is likely to be due to the influence of its composing nucleotides, presumably on the structure of the ribosomal binding site. An analysis of the known sequences of a large number of Escherichia coli genes shows that the use of codons in the second position deviates strongly from the overall codon usage in E. coli. It is proposed that codon selection at the second position is not based on requirements of the gene product (a protein) but is determined by factors governing gene regulation at the initiation step of translation.