Guanidinylated poly(allyl amine) (GA) was synthesized and used as a gene carrier. The degree of guanidinylation in GA increased linearly when the feed ratio of guanidino groups in 1H-pyrazole-1-carboxamidine to amino groups in poly(allyl amine) (PA) was below 0.7, and the amino groups of poly(allyl amine) with a weight-average molecular weight of 15,000 (PA15) were almost replaced with guanidino groups when the ratio reached 2. GA showed good plasmid condensation and protection ability. Nanoparticles with a narrow size distribution, good dispersity, and spherical shape could be assembled between GA and DNA. With an increase in the N/P ratio [where N is the amount of nitrogen in the polycation (for GA, three nitrogens per guanidino group) and P is the amount of plasmid phosphate in the DNA as moles] or the degree of guanidinylation, the zeta-potential of GA/DNA nanoparticles increased, whereas the sizes of GA/DNA nanoparticles decreased sharply with increasing N/P ratios. Compared with polyethylenimine with a weight-average molecular weight of 25,000 and PA15, GA essentially showed decreased cytotoxicity to HeLa, 293T, and HepG2 cell lines, and guanidinylated PA15 exhibited the lowest cytotoxicity. Guanidinylation of PA enhanced its gene transfection. This enhancement was dependent on the degree of guanidinylation and the cell lines. (C) 2009 Wiley Periodicals, Inc. J Appl Polym Sci 112: 926-933, 2009