The cold-induced denaturation of lactate dehydrogenase has been determined in an unfrozen, cryoprotectant free solution at sub-zero temperatures. The cold-induced denaturation temperature (TL) has been found to be -28 degrees C. The results for the first time clearly establish that temperature alone can induce denaturation in a cooled protein solution. The validity of earlier data, obtained in the presence of perturbants (particularly pH or guanidinium chloride), is discussed.