Block copolymers composed of poly(ethylene glycol) with a molecular weight of 2 K and poly(amino acid) random copolymers with 70 mol% L-lysine and 30 mol% L-phenylalanine residues were conjugated with 10, 20 and 30 mol% histidine derivatives at the primary amino end group of the L-lysines. The complex formation of the copolymer and plasmid DNA (pDNA) was confirmed by a gel retardation assay and complete neutralization was achieved at an N/P charge ratio between 1.0 and 2.0. In vitro experiments were carried out on Neuro2A cell lines with an optimized charge ratio of 4.0. The highest transfection efficiency was obtained for the PEG2K-K7-His10/pDNA complex, which decreased gradually with increasing histidine-derivative conjugation ratio to 20 and 30 mol%. The transfection efficiency of the PEG2K-K7-His10 was 5 times higher than that of the PLL homopolymer with a molecular weight of 20.9 K. The cytotoxicity of the complexes was evaluated by a MTT assay with the optimized charge ratio and compared with those obtained from the PLL homopolymer and copolymer without conjugation. The histidine-derivative conjugated copolymers showed almost negligible cytotoxicity to the Neuro2A cell lines, except for the case of PEG2K-K7-His10, which exhibited approximately 70% viability. Introduction of the optimized amount of histidine-derivative into the poly(amino acid) copolymer played an important role in increasing the transfection efficiency.