Formation of pellets by Aspergillus nidulans is primarily due to agglomeration of the fungal conidiospores. Although agglomeration of conidiospores has been known for a long time, its mechanism has not been clearly elucidated. To study the influence of the fungal conidiospore wall hydrophobicity on conidiospore agglomeration, pellet formation of an A. nidulans wild type and strains deleted in the conidiospore-wall-associated hydrophobins DewA and RodA was compared at different pH values. From contact angle measurements, RodA was found to be more important for the surface hydrophobicity than DewA. The absence of either hydrophobin led to a decrease in the relative amount of biomass present as pellets at all pH values as well as a decrease in the average size of the pellets. For all strains, an increasing alkalinity of the medium resulted in an increased pellet formation. Together with measurements of electrophoretic mobility, it is concluded that both the electrical charge and hydrophobicity of the conidiospores affects the pellet formation but that the conidiospore agglomeration process cannot be ascribed to these factors alone.