The half-life of the activity of the PST-01 protease that was secreted by organic solvent-tolerant Pseudomonasaeruginosa PST-01 was very long in the presence of methanol as compared to that in the absence of methanol. The conformational transitions of the PST-01 protease, α-chymotrypsin, thermolysin, and subtilisin in the presence and absence of methanol were monitored by measuring the CD spectra. The conformational stabilities of the PST-01 protease and subtilisin in the presence of methanol were higher than those in the absence of methanol. This resulted in high stability of these proteases in the presence of methanol. Furthermore, it was suggested that the organic solvent stabilities of enzymes were closely related to the secondary structure by monitoring the conformational transitions of polyamino acids, which form the particular conformations, in the presence and absence of methanol.