Cryptococcus neoformans is an excellent model system for studies on the molecular pathogenesis of fungal infections. There is only one dominant selectable market that can be used in the transformation of this organism, and we wanted to develop another. We found that various strains of C. neoformans are very sensitive to the aminoglycoside antibiotic nourseothricin, and that spontaneous resistance to this drug must be an extremely rare event. Resistance to nourseothricin is conferred by the product of the nourseothricin acetyltransferase gene (nat1) from Streptomyces noursei. In order to express this gene in C. neoformans, we created a fusion construct by driving expression of natl with the promoter sequence from a C. neoformans actin gene. Biolistic transformation of the serotype A C. neoformans strain H99 and the serotype D strain JEC21 with this construct resulted in transformation efficiencies of approximately 1,000 transformants microg(-1) of DNA and 20 transformants microg(-1) of DNA, respectively. Southern blots were performed using DNA from some of the H99 transformants, and this confirmed that all of the resistant isolates had the construct integrated in a random fashion within the genome. There was no cross-resistance of the nourseothricin-resistant transformants to hygromycin B, which is the other antibiotic used as a dominant selection marker in C. neoformans. The development of nourseothricin resistance as a second dominant selectable market will be helpful in future molecular studies on this important pathogenic fungus.