The regulator of G protein signaling homolog Crg1 was found to be a key regulator of pheromone-responsive mating in the opportunistic human fungal pathogen Cryptococcus neoformans. A mutation in the CRG1 gene has greatly increased virulence in the prevalently distributed MATalpha strains of the fungus. Mouse survival time was shortened by 40%, and the lethal dosage was 100-fold less than that of wild-type strains. In addition, the increased virulence of crg1 mutant strains was dependent on the transcription factor homolog Ste12alpha but not on the mitogen-activated protein kinase homolog Cpk1. The enhanced mating due to CRG1 mutation, however, was still dependent on Cpk1. Interestingly, crg1 mutants of MATalpha cells produced dark melanin pigment under normally inhibitory conditions, which may relate to the mechanism for increased virulence.