Transcription initiation of the yeast iso-1-cytochrome c gene (CYC-1) occurs in six major clusters at positions +1, +10, +16, +25, +34, and +43. Potential "TATA elements" lie upstream at positions -154, -106, -52, and -22. Analysis of the TATA region suggests that three of these TATA sequences are functional and contribute to initiation at CYC-1, with the -106 TATA promoting initiation at +1, +10, and +16; the -52 TATA, at +16, +25, +34, and +43; and the -22 TATA, at +34 and +43. Deletions changing the spacing between the TATA sequences and the region of transcription initiation do not change the location of the CYC-1 transcription start points. This finding suggests that at least part of the information determining mRNA initiation sites is encoded within the DNA sequence at the site of transcription initiation. Analysis of 18 yeast RNA polymerase II promoters suggests that two classes of DNA sequences serve as preferred sites for transcription initiation. To test this possibility, we replaced some of the normal CYC-1 start sites with one of these sequences, TCGA, and found that transcription initiates at this newly introduced sequence. These results are in contrast to those from higher eukaryotes, where RNA polymerase II typically initiates transcription a fixed distance downstream from the TATA element. The presence of multiple, functional TATA sequences at CYC-1 is inconsistent with the idea that RNA polymerase or another transcription factor attaches to the template at an upstream activation site and scans for the nearest TATA element.