In Ustilago maydis, pathogenic development is controlled by the multiallelic b-mating type locus. b encodes two different homeodomain proteins, bE and bW, that dimerize only when they originate from different alleles. The heterodimer is thought to act as a transcriptional regulator of pathogenicity genes. To address the contribution of individual homeodomains to the function of the hetereodimer, amino acids presumed to be critical for the function of homeodomains were altered by introducing deletions and single amino acid substitutions. This analysis demonstrated that both homeodomains are essential for function of the bE/bW complex. All point mutations affecting conserved amino acids in bW resulted in non-functional proteins. For bE the mutational analysis revealed that certain features of the homeodomain, like base-specific contacts and contacts to the sugar phosphate backbone, contribute to function. However, the structural requirements for function of the bE homeodomain appear to be more flexible than those of the bW homeodomain. We compare our data with the related systems in Schizophyllum commune, Coprinus cinereus and Saccharomyces cerevisiae.