The antifungal antibiotic prumycin, 4-D-alanylamino-2 amino-2,4-dideoxy-L-arabinopyranose, is produced by Bacillus cereus AJ-11252 and B. subtilis AJ-1316. To obtain good prumycin producers, breeding experiments with B. subtilis AJ-1316 were done assuming the biosynthetic pathway via glucosamine. B. subtilis AJ-1316 was selected as a prumycin-producing strain from 100 Bacillus strains. Azaserine-resistant mutants (glutamine analogue-resistant) were derived from AJ-1316, with strains A-11 and A-16 producing 3.0 and 2.8 g/l prumycin respectively (parent AJ-1316 produced 1.3 g/l). Arginine-sensitive mutants derived from A-11 showed higher prumycin production, with strains ll-10 and ll-13 producing 4.6 and 4.4 g/l respectively. It was concluded that prumycin biosynthesis is greatly related to the biosyntheses of glutamic acid, glutamine, and arginine.