The effects of methanol, ethanol, n-propanol, trifluoroethanol, ethylene glycol monobutylether, and 1,4-dioxane on “Kunitz” soybean trypsin inhibitor (STI) was systematically investigated by the circular dichroism (CD) probe and measurement of inhibitory activity. Upon denaturation, the large positive CD band at 226 nm disappears and a new negative CD band at 239 nm is observed at the transition midpoint from native to denatured state. The 239-nm band is assigned to the n − π ∗ transition of the amide bonds of the polypeptide main chain. The commonly observed peak at 245 nm is not a true CD band but is produced by the combination of the positive band at 226 nm and this negative band at 239 nm. The partly denatured STI at the transition midpoint is of a similar aperiodic conformation as native STI and has about 50% of the original inhibitory activity. The unique folding of the polypeptide main chain of the inhibitor is important for its ability to inhibit trypsin. Depending on the conditions, the denaturation of STI by increasing concentrations of organicsolvents can be considered as a three-stage phenomenon: (a) The conformation of the main chain polypeptide backbone is not altered but the side chains are slightly perturbed; (b) the reorganization of the main chain conformation occurs, whereby α-helical and β-pleated sheet structures are formed in most cases; the inhibitory activity decreases concurrently with the reorganization of conformation; (c) at very high concentrations of the perturbant, the pleated sheet structures appear as the predominant conformation, followed by aggregation. The broad and flat negative CD band centered at 275 nm, observed in solutions of denatured STI, probably is contributed chiefly by disulfide chromophores. The vibronic fine structure of the phenylalanine bands is accentuated in denatured STI.