Fks1p and Fks2p are catalytic subunits of beta-1,3-glucan synthase, which synthesize beta-1,3-glucan, a main component of the cell wall in Saccharomyces cerevisiae. Although Fks1p and Fks2p are highly homologous, sharing 88.1% identity, it has been shown that Fks2p is more sensitive than Fks1p to one of echinocandin derivatives, which inhibits beta-1,3-glucan synthase activity. Here we show a similar differential sensitivity between Fks1p and Fks2p to a novel beta-1,3-glucan synthase inhibitor, aerothricin3 [corrected]. To investigate the molecular mechanism of this differential sensitivity, we constructed a series of chimeric genes of FKSs and examined their sensitivity to aerothricin3 [corrected]. As a result, it was shown that a region around the fourth extracellular domain of Fks2p, containing 10 different amino acid residues from those of Fks1p, provided Fks1p aerothricin3 [corrected] sensitivity when the region was replaced with a corresponding region of Fks1p. In order to identify essential amino acid residues responsible for the sensitivity, each of the 10 non-conserved amino acids of Fks1p was substituted into the corresponding amino acid of Fks2p by site-directed mutagenesis. Surprisingly, only one amino acid substitution of Fks1p (K1336I) conferred Fks1p hypersensitivity to aerothricin3 [corrected]. On the other hand, reverse substitution of the corresponding amino acid of Fks2p (I1355K) resulted in loss of hypersensitivity to aerothricin3 [corrected]. These results suggest that the 1355th isoleucine of Fks2p plays a key role in aerothricin3 [corrected] sensitivity.