Liver targeting of plasmid DNA was achieved through conjugation of pullulan derivatives with chelate residues based on metal coordination. Triethylenetetramine (Ti), diethylenetriamine pentaacetic acid (DTPA), and spermine (Sm) were chemically introduced to pullulan, a polysaccharide with an inherent affinity for the liver, to obtain various pullulan-Ti, pullulan-DTPA, and pullulan-Sm derivatives. Irrespective of the type of pullulan derivatives, intravenous injection of the pullulan derivatives-plasmid DNA conjugates with Zn2+ coordination significantly enhanced the level of gene expression only in the liver to a significant greater extent than that of free plasmid DNA. The enhanced gene expression by the pullulan-DTPA-plasmid DNA conjugate was specific to the liver and the level was significantly higher than that of the pullulan-DTPA-plasmid DNA mixture. The level of gene expression depended on the percentage of chelate residue introduced, the mixing ratio of the plasmid DNA-DTPA residue in conjugate preparation, and the plasmid DNA dose. The gene expression induced by the conjugate lasted over 12 days after injection. A fluorescent-microscopic study revealed that the plasmid DNA was localized at the liver after injection of the pullulan-DTPA-plasmid DNA conjugate with Zn2+ coordination. Pre-injection of both arabinogalactan and galactosylated albumin suppressed significantly the liver level of gene expression, in contrast to that of mannosylated albumin, indicating that the plasmid DNA in the conjugate was transfected at hepatocytes. We conclude that the Zn2+-coordinated pullulan conjugation is a promising way to enable the plasmid DNA to target to the liver for gene expression as well as to prolong the time duration of gene expression