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Cationic gold nanoparticles modified with 2-aminoethanethiol, 8-amino-1-octanethiol, and 11-amino-1- undecanethiol were prepared by NaBH4 reduction of HAuCl4 in the presence of thiols in water or a water/ethanol mixture solvent. The formation of gold nanoparticles depended on the kind of solvent, and the concentration of NaBH4, HAuCl4, and thiols. A low concentration of HAuCl4 and a high concentration of NaBH4 facilitated the formation of gold nanoparticles. A high concentration of HAuCl4 solution resulted in aggregation and a low concentration of NaBH4 did not induce the formation of gold nanoparticles. The size of the gold nanoparticles increased with an increase of the concentration of the HAuCl4 solution, while it decreased with an increase of the concentration of the NaBH4 solution. When thiols were present, the HAuCl4/thiol ratio must be in the range of 1:1 to 1:2 for the formation of gold nanoparticles in both the water and water/ethanol mixture solvents. Modification with thiol molecules changed surface charge of the gold nanoparticles from negative to positive, and stabilized the gold nanoparticles. The cationic gold nanoparticles promoted gene transfection. A high ratio of gold nanoparticles to DNA increased transfection efficiency. However, the length of the alkyl groups had no evident effect.