Autonomously replicating sequence (ARS)-binding factor 1 (ABF1) is a multifunctional protein involved in transcriptional activation and repression, as well as DNA replication, in yeast. The ADH1 gene, encoding alcohol dehydrogenase 1, contains two ABF1 consensus binding sites in the promoter and the coding regions. To examine the effect of ABF1 on expression of the ADH1 gene, we constructed an ADH1-lacZ fusion plasmid. Both ABF1 binding sites appeared to be transcriptional activators because deletions and mutations of these sites decreased transcriptional activity. The ABF1 binding sites also acted in an orientation-independent manner when a synthetic ABF1 binding site was inserted into the yeast CYC1 gene lacking its transcriptional activation region. A gel mobility shift assay showed that ABF1 bound in vitro to both ABF1 binding sites in the promoter and coding regions. In a glycerol medium the degree of activation by ABF1 was higher than in a glucose medium. The expression of ADH1 was activated synergistically by both ABF1 binding sites. These observations suggest that ABF1 transactivates the ADH1 gene through its binding sequences in both the promoter and coding regions.