The promoter region of the highly regulated INO1 structural gene of yeast has been investigated. The major transcription initiation start site (+1) was mapped to a position located five nucleotides upstream of the previously identified initiation codon. The INO1 TATA is located at -116 to -111. The INO1 promoter region was used to construct fusions to the Escherichia coli lacZ gene. All INO1 fusion constructs that retained regulation in response to the phospholipid precursors inositol and choline, contained at least one copy of a nine bp repeated element (consensus, 5'-ATGTG-AAAT-3'). The smallest fragment of the INO1 promoter found to activate and regulate transcription of the fusion gene from a heterologous TATA element was 40 nucleotides in length. This fragment contained one copy of the nine bp repeat and spanned the INO1 promoter region from -259 to -219. However, when an oligonucleotide containing the nine bp repeated sequence was inserted 5' to the CYC1 TATA element, it failed to activate transcription.