Most dimorphic fungal pathogens grow as non-pathogenic moulds in soil and convert to pathogenic yeast in the host, suggesting that virulence factors are upregulated during phase transition. Such factors have been difficult to identify. We analysed BAD1 (formerly WI-1), a virulence factor in the dimorphic fungus Blastomyces dermatitidis, for expression in yeast and mycelial morphotypes. BAD1 was expressed in yeast but not in mycelia of North American strains of B. dermatitidis, and this expression pattern was confirmed for BAD1 transcript. BAD1 under the control of its promoter was transferred into African B. dermatitidis lacking a native BAD1 locus, and phase-specific expression was conserved. Sequence similarity was identified between the BAD1 promoter and the promoters of two yeast phase-specific genes in Histoplasma capsulatum. In H. capsulatum BAD1 transformants, yeast phase-specific expression of BAD1 was conserved, and no transcript was detected in mycelia. BAD1 beta-galactosidase reporter fusions analysed in B. dermatitidis and H. capsulatum confirmed that BAD1 is transcriptionally regulated in both fungi. BAD1 promoter activity and surface BAD1 expression were detected 6 h after shifting mycelia to 37 degrees C. Thus, BAD1 is expressed after transition to the pathogenic yeast morphotype and is regulated by a mechanism for phase-specific gene expression that appears to be conserved.