Highly toxic polyallylamine (PA) was reacted with a varying amount of a novel linker, 6-(N,N,N',N'-tetramethylguanidinium chloride) hexanoic acid (Tmg-HA), to prepare a series of tetramethylguanidinium-PA (Tmg-PA) polymers, which were used as vectors for gene transfection. The extent of attachment of the linker, Tmg-HA, to the PA backbone was determined by 2,4,6-trinitrobenzene sulfonic acid assay. The modified polymers (Tmg-PAs), when complexed with pDNA, exhibited good condensation ability. The nanoparticles, so formed, were characterized by their size and zeta potential and were subsequently evaluated for their toxicity and transfection ability on various mammalian cells, viz., HeLa, CHO, and HEK 293 cells. Mobility shift assay revealed that on increasing the percent substitution of Tmg-HA onto PA (from Tmg-PA1 to Tmg-PA6), relatively higher amounts of modified polymers were required to retard the mobility of a fixed amount of DNA. Besides, Tmg-PA polymers provided sufficient protection (ca. 8488%) to bound DNA against nucleases and one of the formulations, Tmg-PA2 (ca. 15% substitution) displayed the highest transfection efficiency outcompeting the commercial transfection reagent, Lipofectamine (TM) with minimal cytotoxicity. More impressively, the transfection efficiency increased despite recording a decrease in the buffering capacity of the grafted polymers suggesting that buffering capacity is not the sole parameter in determining the gene delivery efficiency of a vector system. (c) 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012