Fourier transform (FTIR) difference spectra of pepsin minus diazoacetylnorleucine methyl ester (DAN) or minus diazoacetyl-L-phenylalanine methyl ester (DAP) modified pepsin, respectively, demonstrated that Asp-215 is not deprotonated in pepsin. The FTIR difference spectrum of pepsin minus 1,2-epoxyparanitrophenoxypropane (EPNP) modified pepsin demonstrates that Asp-32 is present in pepsin as CO2- anion. The position of the v(C = O) vibration demonstrates that no (O...H...O)- hydrogen bond between Asp-215 and Asp-32 is formed. Furthermore, no H3O+ is present in the active center. Studies of the complex of pepsin with the inhibitor pepstatin prove that the inhibitor removes the water from the active site and Asp-32 becomes protonated.