Ninety-five streptomycin-nonproducing mutants derived from Streptomyces griseus FT-1 by UV-irradiation could be classified into major two classes by cosynthesis tests. Class I mutants (42 strains) were mutants blocked in the pathway of streptomycin biosynthesis while class II mutants (49 strains) required a factor for streptomycin biosynthesis which was excreted by the parental or class I mutant strains. The factor could be replaced by synthetic A-factor (2S-isocapryloyl-3-S-hydroxymethyl-gamma-butyrolactone) which restored both streptomycin biosynthesis and spore formation in the class II mutants. A-Factor deficient mutants were obtained from several strains of S. griseus and S. bikiniensis at high frequency by treatment with acridine orange or incubation at high temperature. A mutant whose streptomycin biosynthesis was independent of A-factor deficiency was found. The production of A-factor was distributed among various species of actinomycetes.