GCN4 encodes a transcriptional activator in Saccharomyces cerevisiae that is regulated at the translational level. We show that an approximately 240-nucleotide segment from the GCN4 mRNA leader containing four AUG codons is sufficient to confer translational control typical of GCN4 upon a GAL1-lacZ fusion transcript. Regulation of the hybrid transcript is dependent upon multiple positive (GCN) and negative (GCD) trans-acting factors shown to regulate GCN4 expression post-transcriptionally. This result limits the target sequences for these factors to a small internal segment of the GCN4 mRNA leader. The elimination of AUG codons within this segment substantially reduces the usual derepressing effect of mutations in five GCD genes upon GCN4-lacZ expression. This supports the idea that the products of these negative regulatory genes act by modulating the effects of the upstream AUG codons on translation of GCN4 mRNA.