A series of novel cationic lipids based on 1,4,7,10-tetrazacyclododecane (cyclen) with the imidazole group as the pH-sensitive moiety and various aliphatic long chains were designed and synthesized. Cationic liposomes were prepared by mixing the lipids and the helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in an appropriate molar ratio. The liposomes showed good stability and could condense plasmid DNA into nanosized particles (∼100 to ∼250 nm) with a positive zeta-potential (+10-25 mV). CCK-8-based cell viability assays showed a relatively lower cytotoxicity of the lipoplexes compared to commercially available lipofectamine 2000. Both enhanced green fluorescent protein and luciferase assays were carried out to investigate the in vitro transfection efficiency (TE) of the lipoplexes. Results showed that both the structures of the hydrophobic chain and the linking bond significantly affected the TE, and the linoleyl-containing lipoplex gave the best TE, which is comparable to lipofectamine 2000. The imidazole group was demonstrated to play an important role in the transfection, and the imidazole-absent analog gave dramatically lower TE. Furthermore, it was also found that Ca(2+) could largely enhance the TE of these lipids, and the optimized TE was about 5 times higher than lipofectamine 2000. Flow cytometry demonstrates that the enhancement of TE by Ca(2+) was caused by the improvement of cellular uptake. These results suggest that the cyclen-imidazole containing lipids might be promising non-viral gene delivery vectors.