Homogeneous fragments of exosporium were isolated and purified from mature spores of a highly sporogenic mutant derived from Clostridium botulinum type A strain 1901. The exosporium was composed of three lamellae and showed a hexagonal array when negatively stained. The hexagonal array of isolated exosporium was resistant to sodium dodecyl sulfate, urea, dithiothreitol, and proteolytic enzymes such as trypsin, pronase, and nagarse, except for pepsin. The hexagonal array was partially disintegrated with 5 M guanidine-HCl and almost completely disrupted with 8 M urea in combination with 1% mercaptoethanol under alkaline conditions. The purified exosporium fraction was composed mainly of protein (69.1%) and lipids (13.8%). A small amount of amino sugars (2.5%) was present, but neutral sugars could not be detected. The exosporium protein had a predominantly acidic amino acid composition accompanied by low levels of cystine, methionine, and histidine.The spores of some species of Bacillus and Clostridium are known to be loosely enveloped by sac-like exosporium. The fine structure of the exosporia has been well documented using ultrathin sectioning (4, 7, 31), negative staining (12,19,32), and freeze-etching (13,26,32). The morphology of the spores and exosporia of Clostridium botulinum have been demonstrated by electron microscopy to vary according to toxigenic types and strains. The spores of type A are covered by multilayered exosporia (6,28,29), whereas those of type B are enveloped by open-ended, sheathlike exosporia (29). The outstanding features of the spores of type E are the presence of numerous tubular appendages and the delicate fibrillar structure of the membraneous and large exosporia (11,12).The exosporia of Bacillus cereus (7,20) and Bacillus thuringiensis (25) were isolated from spores and examined in detail concerning their chemical composition. In contrast, little information on the morphological and chemical properties of exosporia from the genus Clostridium is available. Recently, Lund et al (18) have isolated exosporium material from a pigmented Clostridium and studied its ultrastructure.In the present study, we isolated a highly sporogenic mutant from the oligosporogenic strain of C. botulinum type A to harvest a sufficient amount of spores. This 443