In an effort to probe the importance of endosomal protonation in pH-sensitive, cationic, lipid-mediated, non-viral gene delivery, we have designed and synthesized a novel cholesterol-based, endosomal pH-sensitive, histidylated, cationic amphiphile (lipid 1), its less pH-sensitive counterpart with an electron-deficient, tosylated histidine head group (lipid 2) as well as a third new cholesterol-based, cationic lipid containing no histidine head group (lipid 3). For all the novel liposomes and lipoplexes, we evaluated hysicochemical characteristics, including lipid:DNA interactions, global surface charge, and sizes. As anticipated, lipid 2 showed lower efficacies than lipid 1 for the transfection of 293T7 cells with the cytoplasmic gene expression vector pT7Luc at lipid:DNA mole ratios of 3.6:1 and 1.8:1; both lipids were greatly inhibited in the presence of Bafilomycin A1. This demonstrates the involvement of imidazole ring protonation in the endosomal escape of DNA. Conversely, endosome escape of DNA with lipid 3 seemed to be independent of endosome acidification. However, with nuclear gene expression systems in 293T7, HepG2, and HeLa cells, the transfection efficacies of lipid 2 at a lipid:DNA mole ratio of 3.6:1 were found to be either equal to or somewhat lower than those of lipids 1 and 3. Interestingly, at a lipid:DNA mole ratio of 1.8:1, lipids 2 and 3 were remarkably more transfection efficient than lipid 1 in both HepG2 and HeLa cells. Mechanistic implications of such contrasting relative transfection profiles are delineated.