alpha-1,3-Glucan is one of the main polysaccharides in the cell wall of filamentous fungi. Aspergillus nidulans has two alpha-1,3-glucan synthase genes, agsA and agsB. We previously revealed that AgsB is a major alpha-1,3-glucan synthase in vegetative hyphae, but the function of AgsA remained unknown because of its low expression level and lack of phenotypic alteration upon gene disruption. To clarify the role of ot-1,3-glucan in hyphal aggregation, we constructed strains overexpressing agsA (agsA(OE)) or agsB (agsB(OE)), in which the other alpha-1,3-glucan synthase gene was disrupted. In liquid culture, the wild-type and agsB(OE) strains formed tightly aggregated hyphal pellets, whereas agsA(OE) hyphae aggregated weakly. We analyzed the chemical properties of cell wall alpha-1,3-glucan from the agsA(OE) and agsB(OE) strains. The peak molecular mass of alpha-1,3-glucan from the agsA(OE) strain (1,480 +/- 80 kDa) was much larger than that from the wild type (147 +/- 52 kDa) and agsB(OE) (372 +/- 47 kDa); however, the peak molecular mass of repeating subunits in alpha-1,3-glucan was almost the same (after Smith degradation: agsA(OE), 41.6 +/- 5.8 kDa; agsB(OE), 38.3 +/- 3.0 kDa). We also analyzed localization of alpha-1,3-glucan in the cell wall of the two strains by fluorescent labeling with alpha-1,3-glucanbinding domain-fused GFP (AGED-GFP). alpha-1,3-Glucan of the agsB(OE )cells was clearly located in the outermost layer, whereas weak labeling was detected in the agsA(OE) cells. However, the agsA(OE) cells treated with beta-1,3-glucanase were clearly labeled with AGBD-GFP. These observations suggest that beta-1,3-glucan covered most of alpha-1,3-glucan synthesized by AgsA, although a small amount of alpha-1,3-glucan was still present in the outer layer. We also constructed a strain with disruption of the amyG gene, which encodes an intracellular alpha-amylase that synthesizes alpha-1,4-glucooligosaccharide as a primer for alpha-1,3-glucan biosynthesis. In this strain, the hyphal pellets and peak molecular mass of alpha-1,3-glucan (94.5 +/- 1.4 kDa) were smaller than in the wild-type strain, and alpha-1,3-glucan was still labeled with AGBD-GFP in the outermost layer. Overall, these results suggest that hyphal pellet formation depends on the molecular mass and spatial localization of alpha-1,3-glucan as well as the amount of alpha-1,3-glucan in the cell wall of A. nidulans.