Novel, double-chained pyridinium compounds have been developed that display highly efficient DNA transfection properties. The transfection efficiency of several of these compounds is enhanced by an order of magnitude, when compared with the transfection efficiency accomplished with the widely used cationic lipid system, lipofectin. Most importantly, the pyridinium compounds were found to be essentially nontoxic toward cells. Using various reporter genes, such as beta-galactosidase and pNEO (a gene construct that renders cells resistent to antibiotic derivatives of neomycin like G418), we demonstrate that the enhanced efficiency relates to the fact that a relative higher number of cells in the population is transfected (approximately 50% in the case of COS cells) by the pyridinium derivatives, whereas the delivery of DNA per cell is also enhanced. Furthermore, application of the pyridinium derivatives shows little cellular preference in their ability to transfect cells. By systematically modifying the structure of the pyridinium amphiphile, i.e., by changing either the headgroup structure or the alkyl chains, some insight was obtained that may lead to unraveling the mechanism of amphiphile-mediated transfection, and thus to protocols that further optimize the carrier properties of the amphiphile. Our results reveal that unsaturated alkyl chains enhance the transfection properties of the pyridinium-based amphiphiles. Preliminary experiments suggest that the structure-dependent improvement of transfection efficiency, when comparing pyridinium derivatives with lipofectin, likely relates to the mechanism of delivery rather than the packaging of the amphiphile/DNA complex.