FT-IR, in combination with residual amino group determination using a fluorescence technique, has been used to investigate the chemical functional groups involved in the cross-linking reaction between glutaraldehyde and gelatin molecules. The results suggest that, at high pH values (i.e., close to the pK(a) of lysine), the cross-linking reaction is mainly governed by the well-known Schiff base formation, whereas at low pH (i.e., when the amino groups of lysine are protonated), the reaction may also involve the -OH groups of hydroxyproline and hydroxylysine, leading to the formation of hemiacetals.