Acidification of vesicular compartments plays an important role in a number of cellular transport processes, including protein secretion, metal cofactor insertion, glycosylation and pH stability. In the present study, we identify and characterize a component of the vesicular proton pump, Vph1p, to determine its role in the virulence of the AIDS-related fungal pathogen Cryptococcus neoformans. Insertional mutagenesis and plasmid rescue were used to identify the VPH1 gene by screening for mutants defective in laccase activity. Disruption of VPH1 resulted in defects in three virulence factors (capsule production, laccase and urease expression), as well as a growth defect at 37 degrees C, but only a small growth reduction at 30 degrees C. These effects were duplicated by the vacuolar (H+)-ATPase inhibitor bafilomycin A1. Furthermore, the vph1 insertional mutant was also avirulent in a mouse meningo-encephalitis model. Complementation of the insertional mutant with wild-type VPH1 resulted in a recovery of virulence factor expression, normal growth at 37 degrees C and restoration of full virulence. These studies establish the importance of the VPH1 gene and vesicular acidification in the virulence of C. neoformans.