There is a growing interest in the use of collagen matrices for tissue engineering. To prevent rapid degradation and to improve their mechanical properties, collagen matrices have been modified using different crosslinking agents. Among the different agents used, water soluble carbodiimides (such as N'-(3-dimethylaminopropyl)-N-ethylcarbodiimide, EDC) in combination with N-hydroxysuccinimide (NHS) are attractive systems, because no additional chemical entities are incorporated in the matrix. EDC/NHS crosslinking leads to amide bond formation between activated carboxyl groups and amine groups. Recently, we proposed that in addition to amide bond formation, ester links are also formed between activated carboxyl groups and hydroxyl groups. This was based on observations we made after development of a new method to quantify concentrations of carboxyl groups of collagen materials before and after crosslinking. The current study is directed to the influence of ester bond crosslinks formed after crosslinking of collagen with EDC/NHS on its physical-chemical and biomechanical properties. Reconstituted dermal bovine collagen patches (RDBC) were used as model material and were crosslinked with EDC/NHS. In one RDBC group, collagen amine groups were blocked with propionaldehyde prior to crosslinking, while in the other group unprocessed RDBC was crosslinked without additional matrix modifications. It was shown that after activation of collagen carboxyl groups with EDC and NHS, amide crosslinks as well as ester crosslinks with collagen hydroxyl groups were formed. It was furthermore demonstrated that the ester crosslinks of EDC/NHS-crosslinked RDBC could be removed by mild hydrolysis affording collagen matrices with improved mechanical properties.