The PIS1 gene encodes a key branchpoint phospholipid biosynthetic enzyme, phosphatidylinositol synthase. The PIS1 promoter contains the unusual feature of three ATG codons (ATGs1, 2, and 3) in-frame with three stop codons, located just before the authentic start codon (ATG4). Using a PIS1(promoter)-lacZ reporter expression system and site-directed mutagenesis, we investigated the role the "upstream" ATG codons play in modulation of PIS1 expression. Of the single codon changes, mutation of the first ATG (ATG1) resulted in the largest increase of the reporter gene PIS1(promoter)-lacZ expression. All combinations of altered upstream ATG codons also resulted in greater reporter expression. Reverse transcription-PCR revealed that at least some PIS1 transcripts include all AUG codons, and their synthesis is probably directed by a second TATA box upstream of the putative TATA box. These results indicate that the multiple upstream AUG codons are present in at least some PIS1 transcripts and negatively impact PIS1 expression.