Present transformation systems for Cryptococcus neoformans depend on complementation of auxotrophic mutants. We have developed a dominant selection system for transformation of wild-type strains of cryptococci in which resistance to the antibiotic hygromycin B is used as the selectable marker. A heterologous fusion gene construct was created by attaching the putative promoter sequence and start site from a cryptococcal actin gene to a truncated hygromycin B phosphotransferase gene from E. coli. Biolistic transformation with this construct resulted in cryptococci resistant to hygromycin B, and transformation efficiencies approached approximately 500 transformants per μg DNA. The construct was found to exist in transformants as both extrachromosomal and integrative forms. The transformants with integrated constructs were stable both in vitro and in vivo, and constructs were recoverable from most transformed cells using a plasmid rescue technique. This is the first dominant selection system for use in C. neoformans, and it should prove useful for molecular studies with this important pathogenic yeast.