The outer coat fraction (OC-Fr) of Bacillus megaterium ATCC 12872 spore was isolated as a resistant residue after alkali extraction, sonic treatment, and pronase digestion of the spore coat preparation, and its backbone structure was determined by chemical analysis to be composed of galactosamine-6-phosphate (GalN-P) polymers with polypeptides and calcium. OC-Fr was not fully solubilized after ordinary acid hydrolysis. OC-Fr was insensitive to all hexosaminidases tested, and moreover, an isolated fragment, a pentamer of GalN-P, was also resistant to lysozyme and hexosaminidases even after N-acetylation, being sensitive to them to some extent after dephosphorylation. Molecular sieving experiments revealed that the outer coat limited the entry of compounds with a molecular weight of more than 2,000. Exchange of the metal on the spore surface also influenced the heat resistance. Spores of OC-Fr-deficient mutants were less resistant but were still much more resistant than the vegetative cells. These results suggest that the outer coat protects the contents of the spore against chemical, physical and enzymatic treatments owing to the chemical structure itself, composed mainly of GalN-P polymers, and the molecular sieving effect.