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The purpose of this research was to evaluate chitosan lactate (CL) of different molecular weights (MWs) as a DNA complexing agent for its efficiency in transfecting COS-1 cells (green monkey fibroblasts) and its effect on cell viability compared with polyethylenimine (PEI), a commercially available cationic polymer. CL and chitosan base dissolved in dilute acetic acid (chitosan acetate, [CA]) of different MWs (20, 45, 200, 460 kDa) and N/P ratios (21, 41, 81, 121, 241) formed complexes with pSV β-galactosidase plasmid DNA. The complexes were characterized by agarose gel electrophoresis and investigated for their ability to transfect COS-1 cells compared with PEI. Additionally, the effect of CL on the viability of COS-1 cells was investigated using 3-(4,5-dimethyliazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The binding of CL/DNA and CA/DNA was dependent on chitosan MWs. The N/P ratio of CL to completely form the complex with the DNA was higher than that of CA. Both CL and CA were comparable in transfection efficiencies at an N/P ratio of 121, but less efficient than PEI (P<.05). The cell viability in the presence of CL and CA at all MWs was over 90%, whereas that of PEI-treated cells was ≈50%. These results suggest the advantage of CL for in vitro gene transfection, with the ease of preparation of polymer/DNA complexes and low cytotoxicity.