Twenty-five kDa polyethylenimine (PEI) is one of the most efficient nonviral gene transfer agents currently applied as a golden standard for in vitro transfection. In this study, novel 25 kDa PEI derivatives with reductively cleavable cystamine periphery (PEI-Cys) were designed to reduce carrier-associated cytotoxicity and to enhance further the transfection activity. The Michael-type conjugate addition of 25 kDa PEI with N-tert-butoxycarbonyl-N'-acryloyl-cystamine (Ac-Cys-(t)Boc) and N-tert-butoxycarbonyl-N'-methacryloyl-cystamine (MAc-Cys-(t)Boc) followed by deprotection readily afforded PEI-Cys derivatives, denoted as PEI-(Cys)x(Ac) and PEI-(Cys)x(MAc), with degree of substitution (DS) ranging from 14 to 34 and 13 to 38, respectively. All PEI-Cys derivatives had higher buffer capacity than the parent 25 kDa PEI (21.2 to 23.1% versus 15.1%). Gel retardation and ethidium bromide exclusion assays showed that cystamine modification resulted in largely enhanced interactions with DNA. PEI-(Cys)x(Ac) could condense DNA into small-sized particles of 80-90 nm at and above an N/P ratio of 5/1, which were smaller than polyplexes of 25 kDa PEI (100-130 nm). In comparison, PEI-(Cys)x(MAc) condensed DNA into somewhat larger particles (100-180 nm at N/P ratios from 30/1 to 5/1). Gel retardation and dynamic light scattering (DLS) measurements showed that PEI-Cys polyplexes were quickly unpacked to release DNA in response to 10 mM dithiothreitol (DTT). These PEI-Cys derivatives revealed markedly decreased cytotoxicity as compared with 25 kDa PEI with IC(50) values of >100 mg/L and 50-75 mg/L for HeLa and 293T cells, respectively (corresponding IC(50) data of 25 kDa PEI are ca. 11 and 3 mg/L). The in vitro transfection experiments in HeLa and 293T cells using pGL3 as a reporter gene showed that gene transfection activity of PEI-Cys derivatives decreased with increasing DS and PEI-(Cys)x(MAc) exhibited higher transfection activity than PEI-(Cys)x(Ac) at similar DS. Notably, polyplexes of PEI-(Cys)14(Ac) and PEI-(Cys)13(MAc) showed significantly enhanced gene transfection efficiency (up to 4.1-fold) as compared with 25 kDa PEI formulation at an N/P ratio of 10/1 in both serum-free and 10% serum-containing conditions. The modification of PEI with reductively cleavable periphery appears to be a potential approach to develop safer and more efficient nonviral gene vectors.