Both mycobacillin production and sporulation are initiated in the postlog phase of growth of B. subtilis B₃, reaching their maximum by about the same time. Mycobacillin is released during enndotropic sporulation of vegetative cells though not during germination of spores in complex growth medium. Antisporogenic chemicals like glucose (in excess), diethylmalonate, acriflavin, fluoroacetic acid, sodium bisulphite, β-phenethyl alcohol, α-picolinic acid and m-tyrosine inhibit mycobacillin production. By acriflavin and actinomycin D treatment, two types of mutants, oligosporous and asporogenous, were obtained. Mycobacillin production is affected adversely in oligosporous mutants whereas asporogenous mutants do not produce the antibiotic at all.