Altogether 16 cholesterol-based amphiphiles were synthesized. In the first and second groups, nonionic oligo(ethylene glycol) appendages were covalently introduced either indirectly via a succinate spacer or directly respectively to the 3 beta -OH group of cholesterol. A third group of derivatives was prepared where the location of the cationic charge was conserved although the headgroup residue was progressively modified with increasing lengths of oligo(oxyethylene) units. In the fourth group, a cationic center was introduced onto the steroid backbone via increasing lengths of oligo(oxyethylene) spacers. In the fifth group, the poly(oxyethylene) segments of one amphiphile each of series 3 and 4 were replaced by the same length of polymethylene chain. Vesicle formation from the aqueous suspensions of these compounds was confirmed by TEM and dye entrapment. Fluorescence anisotropy and XRD studies revealed remarkable control of membrane characteristics by both the length and location of the oxyethylene segment.