Objective To select the best glutaraldehyde cross-linkage by implantation experiment and obtaining the optimizing acellular dermal matrix(ADM) by the method of NaOH maceration and the best glutaraldehyde cross-linkage. Fibroblasts(FBs) were cultured on optimized ADM and observed to find a good biocompatibility. Methods We prepared rat ADM by the method of NaOH maceration. The resulting ADM was divided into four groups by different cross-linkage, including group A(0min), group B(10min), group C(15min) and group D(30min). We compared physical character, area after implantation, inflammation reaction and the number of infiltrated FBs and blood vessels of four groups by ordinary and histological observation in order to select the best cross-linkage. The cytotoxicity of ADM was evaluated by methyl thiazolyl tetrazolium (MTT) colorimetry. FBs were cultured on optimized ADM and observed by immunohistochemistry and transimission electron microscope(TME). The expression of type Ⅰand Ⅲ precollagen mRNA in the FBs was examined by RT-PCR. Results Four weeks after implantation, areas of uncross-linked ADM were decreased and the ADM closely adhered to the tissue around, inflammation cells could be observed. There were a few inflammation cells and statistical negative correlation between the number of infiltrated cells/blood vessels and cross-linkage in the crosslinked ADM. The best cross-linkage time was 10 min. The cytotoxicity scores of optimized ADM were grade 0 or 1. FBs grew and proliferated well in the optimized ADM. Compared with the one grown on the plate, the expression of type Ⅰand Ⅲ precollagen mRNA of FBs grown in the optimized ADM were decreased. Conclusion The process of NaOH maceration is simple and low cost. The optimized ADM for 10minutes has good biocompatibility and may provide space and transduct information for cells growth. It effers a molecular biology foundation which may reduce scar formation because of lowly regulating m