An asporogenic mutant ofClostridium botulinumtype E, ATCC 9564, was recovered on liver veal agar, which had been supplemented with MnSO4(0.13 mM) and mercaptoacetate (0.2%). Growth in TPG measured by optical density increased exponentially for the two strains during the first 11 h. But subsequently, the optical density of the wild type rapidly declined while the sp−mutant showed a more gradual decrease. The pH decreased significantly during growth but remained unaltered at pH 5–6 during sporulation. The DPA synthesis in the sp+strain paralleled the appearance of refractile spores, while the sp−strain showed no measurable amounts of DPA. Protease and antibiotic activities were detected late in the log growth phase of the wild type. Gas–solid chromatography showed that during growth and sporulation the mercaptoethanol and CO2content declined significantly in the wild type, but remained unchanged in the sp−mutant, whereas N2O was evolved concurrently with sporulation. This study has provided us with an effective method for the isolation of asporogenic mutants ofC.botulinum, as well as a basis for the analysis of the events which occur during the stages of sporulation.