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Four molecular weights of poly-l-lysine (PLL) [1000–5000 Da, 1500–8000 Da, 4000–15,000 Da and 15,000–30,000 Da] and three molecular weights of polyethyleneimine (PEI) [800 Da, 2,000 Da, and 25,000 Da] were used to systematically study the effect of calcium (Ca2+) to improve transfection efficiency of polyelectrolyte complexes. Complexes made using different molecular weights of PLL or PEI polymer showed clear differences in the levels of gene expression in the presence and absence of calcium chloride when tested using A549 human lung carcinoma cells. Complexes formed from PLL or PEI 800 Da were exhibited negligible expression of pDNA according to a luciferase reporter assay. Low molecular weight PLL and PEI 800 Da, however, became highly efficient gene delivery vehicles when calcium was added to the nascent complexes while maintaining the low cytotoxicity of low molecular weight polyamines. Additional analyses indicated that the most effective formulations utilized a threshold level of calcium, which created small, stable particles, but also facilitated unpackaging of the gene complexes and release of pDNA.