Milbemycin antibiotics are produced by Streptomyces hygroscopicus subsp. aureolacrimosus and a newly isolated Streptomyces bingchenggensis, respectively. According to the biosynthetic pathway of milbemycins generated by S. hygroscopicus subsp. aureolacrimosus, a rational screening procedure with UV irradiation and N-methyl-N 0 -nitroso-N-nitrosoguanidine (NTG) mutation was performed to obtain high milbemycin-producing S. bingchenggensis. Aminoacetic acid (Glycine)-resistant mutants (AA r ), propionate-resistant mutants (PRP r ), streptomycin-resistant mutants (STR r ) and 2-deoxy-D-glucoseresistant mutants (DOG r ) were selected successively. A strain S. bingchenggensis BC-109-6 with AA r , PRPr, STR r and DOG r was obtained and its production of milbemycin A3 and A4 reached 1,450 lg/ml, which was 80% higher than that of the ancestral strain S. bingchenggensis BC-101-4. The subculture experiments indicated that the hereditary characteristic of high productivity of S. bingchenggensis BC-109-6 was stable. The production of milbemycin A3 and A4 by S. bingchenggensis BC-109-6 in a 50-l fermentor could reach 1,380 lg/ml after 360 h batch fermentation.