We analyzed 71 clinical and environmental Cryptococcus gattii strains that had been isolated before or after the advent of azole antifungals to determine their level of heteroresistance to fluconazole (LHF). All strains of C. gattii manifested heteroresistance, with LHFs that ranged between 4 microg/ml and 32 microg/ml. A considerably higher proportion of the C. gattii strains (86%) than Cryptococcus neoformans strains (46%) exhibited LHFs that were > or =16 microg/ml. No significant correlation was observed between the molecular type or serotypes of strains and their respective LHF. The strains which expressed a higher LHF were also more resistant to xenobiotics than the strains with a low LHF, and the level of resistance to xenobiotics was significantly higher than that reported for C. neoformans. The heteroresistant subpopulation, whose level of drug resistance had been raised in a stepwise manner to 64 microg/ml, reverted to the original LHF upon daily transfers in drug-free medium. Importantly, the strains with high LHFs were significantly more virulent than those with low LHFs. Since all the clinical isolates that had not been exposed to azole drugs as well as the environmental strains manifested heteroresistance to fluconazole, heteroresistance of C. gattii to azoles is an intrinsic mechanism as in C. neoformans and is associated with the strain's virulence.