In Aspergillus nidulans, the transcriptional activator AlcR mediates specific induction of a number of alc genes. The AlcR DNA-binding domain is a zinc binuclear cluster that differs from the other members of the Zn2Cys6 family in several respects. Of these, the most remarkable is its ability to bind in vitro as a monomer to single sites, whereas only repeated sites (direct or inverted) are necessary and functional in vivo. Deletion of the first five amino acids (following the N-terminal methionine) upstream of the AlcR zinc cluster or mutation of a single residue, Arg-6, impairs the AlcR in vitro binding mainly to symmetrical sites. In vivo, the same mutations result in the inability of A. nidulans to grow on ethanol. The alc- phenotype results from a drastic decrease in activation of its own transcription and, in addition, that of the two structural genes, alcA and aldA, required for ethanol oxidation. This defect seems to be correlated to the inability of the Arg-6 AlcR mutant protein to bind to AlcR palindrome targets, which are essential in the three alc promoters. AlcR shows a unique pattern of binding and of transactivation among the Zn2Cys6 family.