A microbiological assay of vitamins in the sample containing Leucomycin needs to be considered. The growth of Lactobacilli, usually used organisms for microbiological assay, was completely inhibited by Leucomycin in low concentrations. The Leucomycin resistant strains were isolated by means of successive transfer in the medium containing graded amount of Leucomycin. The growth of resistant strain was not inhibited by the presence of 2000μg/ml of Leucomycin and the pantothenic acid and nicotinic acid requirement of resistant strain of Lactobacillus arabinosus 17-5 and the folic acid requirement of resistant strain of Streptococcus faecalis R were the same with that of each parent strain. The standard curves for these vitamins with resistant strains were not influenced even by the presence of a about 500μg/ml Leucomycin.