The inability to recover monomeric protein by heat treatment following the incubation of formaldehyde-treated proteins in ethanol may result from a combination of cross-link formation and a change in protein conformation. Thus, we examined the structural properties of RNase A treated with formaldehyde and ethanol using circular dichroism (CD) spectroscopy. The far-UV spectrum is sensitive to the secondary structure of the protein. Fixation in 10% formalin for up to 1 week did not significantly alter the secondary structure of RNase A relative to theuntreated protein. Additionally, native, unfixedRNase incubated in 100% ethanol for 1 week rapidly reverted back to as native structure after the ethanol was removed.