The filamentous fungus, Aspergillus nidulans, genome contains at least five chitin synthase-encoding genes. chsB is essential for normal hyphal growth. chsA and chsC are likely to be cooperatively required for hyphal wall integrity. In this study, we constructed chsA chsB and chsC chsB double mutants, in which chsB expression was under a repressible promoter [ alcA(p)]. While chsA or chsC single mutants did not show obvious growth defects, the chsA chsB and chsC chsB double mutants showed different phenotypes from the chsB single mutant and from each other under alcA(p)-repressing conditions. The chsA chsB double mutant produced fewer aerial hyphae and the chsC chsB double mutant showed reduced cell mass. These observations support the idea that chsA and chsC each play a different role in hyphal morphogenesis. In addition, the chitin contents of these double mutants were higher than those of the chsB single mutant. When chsA was expressed ectopically under the chsB promoter in the chsB mutant, the growth defects caused by chsB repression were not remedied at all, although an increased level of chsA mRNA was observed. Thus, it is suggested that the gene products of chsA and chsB themselves have different functions in hyphal morphogenesis.