Blastomyces dermatitidis is a primary fungal pathogen of man and other mammals, but like many other human fungal pathogens, relatively little is known about the factors that account for its virulence and pathogenicity. We developed a transformation system to facilitate molecular genetic studies of putative virulence factors from B. dermatitidis. Transformation of the multinucleate yeasts was achieved by electroporation of DNAs containing a dominant selectable marker, hygromycin B (HygB) resistance. Southern analysis showed that transforming DNA invariably integrated ectopically into the chromosome. No evidence was found for extrachromosomal DNA. The HygB resistance could be expressed by either a 375-bp promoter fragment of the B. dermatitidis WI-1 gene encoding adhesin or an Aspergillus gpdA promoter placed 5' of the E. coli hph gene. Primer extension analysis showed that for plasmids containing the WI-1 promoter, transcription of the hph gene initiated within the 375-bp WI-1 promoter fragment. The combination of gene transfer and two promoters capable of independent transcription will allow us to restore or augment gene expression in appropriate strains and test an influence on virulence. Molecular genetic manipulation of B. dermatitidis represents a major advance in our ability to investigate the pathogenesis of blastomycosis and other similar fungal diseases.