Scaffold-guided tissue engineering based on synthetic and natural occurring polymers has gained many interests in recent year. In this study, the development of a chitosan-heparin artificial extracellular matrix (AECM) is reported. Three-dimensional, macroporous composite AECMs composed of heparin (Hep) and chitosan (Chito) were prepared by an interpolyelectrolyte complex/lyophilization method. The Chito-Hep composite AECMs were respectively, crosslinked with glutaraldehyde, as well as cocrosslinked with N,N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide (EDC/NHS) and N-hydroxysuccinimide (NHS). The crosslinking reactions were examined by FT-IR analysis. In physiological buffer solution (PBS), the EDC/NHS-crosslinked Chito-Hep composite AECM showed a relative lower water retention ratio than its glutaraldehyde-crosslinked counterparts. The EDC/NHS-crosslinked Chito-Hep composite AECMs showed excellent biocompatibility, according to the results of the in vitro cytotoxic test. This result suggested that the EDC/NHS-crosslinked Chito-Hep composite AECMs might be a potential biomaterial for scaffold-guided tissue engineering applications. (c) 2008 Wiley Periodicals, Inc. J Appl Polym Sci 109: 3639-3644, 2008.