Fourier-transform infrared (FTIR) spectroscopy has been used to quantitatively examine the secondary structure of imprinted (i.e., lyophilized in the presence of multifunctional ligands followed by removal of the latter) proteins in anhydrous media. Lysozyme, chymotrypsinogen, and bovine serum albumin, imprinted with L-malic acid, all exhibited significant differences in the secondary structure compared to that of their nonimprinted counterparts. A rise in the beta-sheet content, which invariably occurs upon lyophilization, is substantially lower for imprinted proteins. Alterations in the alpha-helix contents of these proteins have also been observed upon imprinting, although these changes are specific to the protein. A structural explanation has been obtained herein for other previously observed aspects of the protein imprinting phenomenon, including the effects of the ligand and the solvent and the lack of enantioselectivity. Exposure to aqueous solution, but not to anhydrous solvents, results in the disappearance of imprinting-induced changes in the secondary structure of proteins.