A large number of strains of Alternaria kikuchiana that cause the black spot disease on pear were isolated from some orchards of Tottori Prefecture, Japan. From them seven strains, which showed different sensitivities to polyoxin B, were selected and used in the experiments. Polyoxin B, at a concentration of 10μM, inhibited more than 50% of the incorporation of glucosamine-¹⁴C into cell-wall chitin in washed mycelia of the polyoxin-sensitive strains and at the same time resulted in an unusually increased accumulation of UDP-N-acetylglucosamine-¹⁴C, which is a precursor of chitin biosynthesis. Both the inhibition of glucosamine-¹⁴C incorporation into chitin and the increase of accumulation of UDP-N-acetylglucosamine-¹⁴C caused by polyoxin B were moderately lowered with decreasing the sensitivity to the antibiotic of the different strains. Crude preparations of chitin synthetase were obtained from these strains. Polyoxin B strongly inhibited all the enzyme preparations in competition with their substrate UDP-N-acetyl-glucosamine. The Km values for the substrate or the Ki values for the antibiotic determined in the enzyme reactions differed slightly from each other. These results indicate that polyoxin B acts as an competitive inhibitor of chitin synthetase, which is concerned in the synthesis of cell-wall chitin of A. kikuchiana. And they also suggest that the polyoxin-resistance of this fungus is caused by a lowered penetration of the antibiotic through the cell membrane into the enzyme site.