The expression of the Saccharomyces cerevisiae CYC1 gene, which encodes iso-1-cytochrome c, produces a family of messenger RNAs whose 5' ends map in the region from position +7 to -93 relative to the first nucleotide at position +1 of the protein-coding DNA sequence. The mechanism of transcription initiation of the CYC1 gene has been examined by using linker-scanning deletions and gene fusions. The various CYC1 derivatives with mutations in the 5' non-coding region were constructed, reintroduced into yeast using a multicopy plasmid, and the mRNA starts mapped by primer extension. The results indicate that four, and possibly five T-A-T-A sequences are located within the 5' non-coding region of the CYC1 gene, and that each T-A-T-A is required for a specific subset of mRNA starts. This conclusion has been confirmed by oligonucleotide mutagenesis of a chromosomal CYC1 T-A-T-A sequence. A loose spatial relationship also exists between the T-A-T-A sequences and the mRNA start sites, and this distance relationship varies from 100 to 60 base-pairs (+/- 15 base-pairs).