In the phytopathogenic fungus Ustilago maydis, sexual and pathogenic development are controlled by the multiallelic b mating type locus. The b locus encodes a pair of unrelated homeodomain proteins termed bE and bW that form a heterodimeric complex when both proteins originate from different alleles. The heterodimer is presumed to be the central regulator for pathogenicity genes. Here, we show that a translational fusion protein comprising specific domains of bE1 and bW2 remains biologically active and binds to a sequence motif in the promoter of lga2, a gene located in the a mating type locus. This b binding sequence 1 (bbs1) is also recognized by the native bE1-bW2 heterodimer in vivo and mediates the b-dependent regulation of the lga2 gene. Our data demonstrate that the bE-bW heterodimer can act as a positive transcriptional regulator.