The roles of the Gbetagamma subunits of the opportunistic human pathogen Aspergillus fumigatus were investigated. The predicted AfuSfaD (Gbeta) protein consists of 353 amino acids and shows 94-98% similarity with other Aspergillus Gbeta subunits. AfuGpgA consists of 90 amino acids showing 95-98% identity with other fungal G-protein gamma subunits. The deletion (Delta) of AfusfaD or AfugpgA resulted in severe impairment in vegetative growth, conidial germination and conidial trehalose breakdown. While the total number of conidia produced by DeltaAfusfaD and DeltaAfugpgA strains on solid medium was only about 1% of wild type, the growth-adjusted conidiation levels were twofold higher than those of wild type. Enhanced formation of conidiophores and elevated AfubrlA mRNA levels were observable in DeltaAfusfaD or DeltaAfugpgA strains in liquid submerged culture. Moreover, overexpression of AfusfaD or AfugpgA caused reduced levels of submerged culture conidiation, indicating that Gbetagamma is involved in negative regulation of conidiation. Gliotoxin and other metabolites were not detected in the chloroform extracts of DeltaAfusfaD and DeltaAfugpgA culture filtrates. Northern blot analyses revealed that, while AfulaeA mRNA levels unchanged, accumulation of gliZ mRNA was delayed by DeltaAfusfaD or DeltaAfugpgA. A model summarizing the roles of AfusfaD and AfugpgA in A. fumigatus is presented.