Six new ether functionalized gemini pyridinium amphiphiles have been synthesized having dodecyl, tetradecyl allcyl chain lengths and three different spacers (i.e. -(CH2)(n)-, where n is 4, 5 and 6) and investigated for their self-assembling behavior by state of the art techniques such as tensiometry, conductivity and spectrofluorometry. These new pyridinium gemini surfactants exhibit lower cmc values as compared to other gemini surfactants reported in literature. These amphiphiles form stable complexes with DNA as established by agarose gel electrophoresis and ethidium bromide exclusion experiments. MIT (3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyitetrazolium bromide) assay was carried out in vitro on C6 glioma cell line for cytotoxicity assessment of new pyridinium geminis. The dynamic light scattering (DLS) and transmission electron microscopy (TEM) have been used to measure the micellar size of gemini surfactants. Further, thermal stability of these amphiphiles has been evaluated by thermogravimetric analysis (TGA). The dependence of self-assembly behavior and other properties on spacer as well as alkyl chain length has been established. (C) 2013 Elsevier Inc. All rights reserved.