Previous studies have demonstrated an important role for the vacuole in the virulence of the fungus Cryptococcus and studies in yeast have implicated the vacuolar protein Vps41 in copper loading of proteins such as iron transporters. However, our studies found that a cryptococcal vps41Delta strain displayed wild-type growth on media containing iron and copper chelators and normal activity of the copper-containing virulence factor laccase as well as almost normal growth at 37 degrees C and wild-type production of the virulence factor capsule. Despite these attributes, the vps41Delta mutant strain showed a dramatic attenuation of virulence in mice and co-incubation of mutant cells with the macrophage cell line, J774.16, resulted in a dramatic loss in viability of the vps41Delta mutant strain at 10 h compared with wild-type and complemented strains. Closer examination revealed that the vps41Delta mutant displayed a dramatic loss in viability after nutrient starvation which was traced to a failure to undergo G2 arrest, but there was no defect in the formation of autophagic or proteolytic vesicles. Our results indicate that VPS41 plays a key role in regulating starvation response in this pathogenic organism and that defects in cell cycle arrest are associated with attenuated pathogenic fitness in mammalian hosts.