The transcription factor Haa1 is the main player in reprogramming yeast genomic expression in response to acetic acid stress. Mapping of the promoter region of one of the Haa1-activated genes, TPO3, allowed the identification of an acetic acid responsive element (ACRE) to which Haa1 binds in vivo. The in silico analysis of the promoter regions of the genes of the Haa1-regulon led to the identification of an Haa1-responsive element (HRE) 5'-GNN(G/C)(A/C)(A/G)G(A/G/C)G-3'. Using surface plasmon resonance experiments and electrophoretic mobility shift assays it is demonstrated that Haa1 interacts with high affinity (K(D) of 2 nM) with the HRE motif present in the ACRE region of TPO3 promoter. No significant interaction was found between Haa1 and HRE motifs having adenine nucleotides at positions 6 and 8 (K(D) of 396 and 6780 nM, respectively) suggesting that Haa1p does not recognize these motifs in vivo. A lower affinity of Haa1 toward HRE motifs having mutations in the guanine nucleotides at position 7 and 9 (K(D) of 21 and 119 nM, respectively) was also observed. Altogether, the results obtained indicate that the minimal functional binding site of Haa1 is 5'-(G/C)(A/C)GG(G/C)G-3'. The Haa1-dependent transcriptional regulatory network active in yeast response to acetic acid stress is proposed.