Abstract: AIMThere are many factors affecting hybridization in cDNA chip: DNA concentration immobilized on glass surface, spotting solution dissolving DNA, the concentration and purity of fluorescence-labeled probe, hybridization solution, hybridization temperature, hybridization time, Cy3- and Cy5-labeled probes, etc. In order to improve hybridization efficiency, tests were designed to optimize these factors. METHODSFactors are changed one by one. Optimal values are selected by comparing those of different groups. RESULTS AND CONCLUSIONThe efficient hybridization condition is as follows: 0.50 μg·μL^-1 DNA fragments resolved in 1×Micro Spotting Solution (ArrayIt^TM) hybridize with purified fluorescent probe in 0.5% SDS / 10×SSC at 42℃.