Transcription of the PHO84 gene encoding a Pi transporter in Saccharomyces cerevisiae is regulated by the Pi concentration in the medium. The promoter region of PHO84 bears five copies of the motif 5′-CACGT(G/T)-3′, a candidate for the upstream activation site (UAS) that binds the transcriptional activator protein of the phosphatase regulon, Pho4p. These motifs are found at nucleotides - 880 (site A), —587 (B), - 436 (C), - 414 (D), and - 262 (E) relative to the putative ATG codon of PHO84. The Pho4p protein binds to all five 6-bp motifs with various affinities. Deletion analysis of the PHO84 promoter using a PHO84-lacZ fusion gene and base substitutions in the 6-bp motif revealed that two copies of the 6-bp motif, either C or D, and E, are necessary and sufficient for full regulation of the PHO84 gene. Results of expression studies with a CYC1-lacZ fusion gene with various 36-bp oligonucleotides including the 30-bp sequences around site D or E, or with modified sequences, inserted in the CYC1 promoter region indicated that the 6-bp motif flanked by a thymine nucleotide at its 5′ end is much less effective as a UAS site for Pho4p in vivo than other versions. Thus, the consensus sequences for phosphatase regulation are 5′-GCACGTGGG-3′ and 5′-GCACGTTTT-3′ which differ from the binding sequences for the Cpflp protein required for transcription of the genes in methionine biosynthesis and for centromere function. However, Pho4p binding in vitro was unaffected by modification of the 5′ or 3' flanking sites of the 6-bp motif, while modification inside the 6-bp motif affected it severely. The UAS function of the GCACGTTTT motif with respect to the Pi signal depends on its orientation in the promoter sequence.