We have utilized a recombinant vector system designed to study gene control elements by fusing them to the Escherichia coli galactokinase gene (galK) to examine the effects of gene expression that result from introducing changes into the 5' noncoding leader region of the galK transcription unit. We varied the length of the 5' leader region and found no effect on galK expression, provided that the galK initiation codon remained the first AUG in the transcription unit. Using synthetic linkers, we then inserted specific sequences, each containing a single AUG codon at a defined position within the leader region. We found that the AUG inserts had widely different effects on galK expression and that the sequences surrounding the inserted AUG codons determined the magnitude of these effects. In addition, we placed these upstream inserted AUG codons into each of the three possible translation reading frames so that translation occurring in these frames terminates prior to, within, or downstream of the galK initiation codon, respectively. Single-nucleotide frameshift mutations were also introduced into one of these constructs to shift upstream translation into the other two possible reading frames. Depending upon where upstream translation stopped relative to the galK initiation codon, we observed consistently different effects on galK expression. Our results show that an upstream AUG that interferes with downstream translation initiation exerts its greatest effect when it translates out-of-frame through the downstream initiator into the gene. If translation is stopped upstream of or within the initiator, an unexpectedly high level of expression from the downstream AUG is maintained.