Three series of pyridinium cationic lipids useful as nonviral gene delivery agents were prepared by reaction of pyrylium salts with aminodiols, followed by acylation with fatty acyl chlorides. On the basis of this set of compounds, we undertook a comprehensive structure-activity relationship study at the level of the linker, hydrophobic anchor, and counterion in order to identify the structural elements that generate the highest transfection efficiency for this new type of cationic lipid. The results revealed that when formulated with cholesterol at a 1:1 molar ratio, the 1-(1,3-dimyristoyloxyprop-2-yl)-2,4,6-trimethylpyridinium, under the form of hexafluorophosphate (5AMyr) or chloride (5DMyr), was able to transfect NCI-H23 lung carcinoma with efficiencies surpassing classic DOTAP-based formulations and with lower cytotoxicity. Subsequent tests on other malignancies yielded similarly promising results.