The pathogenic yeast Cryptococcus neoformans (Cn) var. gattii causes meningoencephalitis in healthy individuals, unlike the better known Cn varieties grubii and neoformans, which are common in immunocompromised individuals. The virulence determinants and mechanisms of host predilection are poorly defined for var. gattii. The present study focused on the characterization of a Cu,Zn superoxide dismutase (SOD1) gene knock-out mutant constructed by developing a DNA transformation system. The sod1 mutant was highly sensitive to the redox cycling agent menadione, and showed fragmentation of the large vacuole in the cytoplasm, but no other defects were seen in growth, capsule synthesis, mating, sporulation, stationary phase survival or auxotrophies for sulphur-containing amino acids. The sod1 mutant was markedly attenuated in virulence in a mouse model, and it was significantly susceptible to in vitro killing by human neutrophils (PMNs). The deletion of SOD1 also resulted in defects in the expression of a number of virulence factors, i.e. laccase, urease and phospholipase. Complementation of the sod1 mutant with SOD1 resulted in recovery of virulence factor expression and menadione resistance, and in restoration of virulence. Overall, these results suggest that the antioxidant function of Cu,Zn SOD is critical for the pathogenesis of the fungus, but is dispensable in its saprobic life. This report constitutes the first instance in which superoxide dismutase has been directly implicated in the virulence of a fungal pathogen.