Cryptococcus neoformans is an opportunistic fungal pathogen that synthesizes and catabolizes inositol. This study demonstrates inositol synthesis from glucose-6-phosphate via inositol-1-phosphate synthase and catabolism to glucuronic acid via inositol oxygenase in this organism. These inositol synthetic and catabolic pathways are regulated in opposition; repressing conditions for one are inducing conditions for the other. An inositol-requiring strain was generated by UV mutagenesis. Without inositol, this mutant strain undergoes 'inositol-less' death, during which time the phosphatidylinositol composition of the membranes decreases without alteration of the proportion of other phospholipids. The mutation on this strain results in no detectable inositol synthetic activity but normal (wild-type) inositol catabolic activity. This inositol-requiring mutant strain reverted at a high frequency. Classical genetic experiments revealed that the majority of the reverting mutations are at second sites. Interestingly, the revertants exhibited unusual morphological phenotypes when deprived of inositol, while provision of inositol restored wild-type morphology. Inositol metabolism is clearly important for growth and development of C. neoformans and may be involved in this organism's mechanism for survival as both a saprophyte in soil and a parasite in humans.