Protoplasts of Bacillus stearothermophilus NCA 1503-4R are resistant to osmotic rupture and are not sensitive to mechanical manipulation. Protoplast stability is maintained by divalent cations. The thermostability of protoplasts is enhanced when the cells are grown at elevated temperatures. The membrane content of the cell and the protein-to-lipid ratio of the membrane increases as the growth temperature is increased. The membrane-bound nicotinamide adenine dinucleotide (reduced form) oxidase system from cultures grown at 70 C was more thermostable than the same enzyme system from cultures grown at 55 C. Alkaline phosphatase was resistant to thermal inactivation in the intact protoplast. The extent of this protection is dependent on protoplast stability.