While RNA polymerase II of the yeast Saccharomyces cerevisiae initiates transcription at discrete sites, these sites are located over a wide range of distances from the TATA box for different genes. This variability has led to a number of proposals for consensus sequences located at the initiation site which, in conjunction with the TATA box, would direct initiation. We tested this hypothesis via oligonucleotide-directed mutagenesis, by placing the sequence CAAG, a member of one of these consensus sequences, upstream of the coding sequence of the CYC7 gene at a site at which initiation does not occur. The distance between the TATA sequence and this putative initiation site was varied by inserting it into the wild-type gene and three deletion mutants. The results demonstrated that this sequence can serve as an initiation site when located 49, 77, or 106 bp from the TATA sequence, but not when located 30 bp away.