Monomers of some amphiphiles organize into bilayers to form liposomes and niosomes. Such bilayers are unstable or leaky and hence cholesterol is a common ingredient included to stabilize them. Cholesterol stabilizes bilayers, prevents leakiness, and retards permeation of solutes enclosed in the aqueous core of these vesicles. Other than cholesterol a material with good bilayer-stabilizing properties is yet to be identified. We have substituted cholesterol with fatty alcohols in niosomes containing polyglyceryl-3-di-isostearate (PGDS) and polysorbate-80 (PS-80) to explore their membrane-stabilizing property via permeation studies. Niosomes of polyglyceryl-3-di-isostearate, fatty alcohol/cholesterol, and polysorbate were prepared by ether injection method. Aqueous solution of ketorolac tromethamine (KT) was entrapped in them. The effects of alkyl chain length of fatty alcohols (C(12), C(14), C(16), C(18), and C(16+18)), of acyl chain length of polyoxyethylene sorbitan monoester surfactants, and of the molar ratio of lipid mixture on the release rate of ketorolac from niosomes were assessed by employing modified dissolution-dialysis method. Niosomes with cholesterol or fatty alcohols have exhibited a common release pattern. Niosomes containing fatty alcohol showed a considerably slower release rate of KT than those containing cholesterol. Based on the release rate, fatty alcohols can be ranked as stearyl<myristyl<cetyl<lauryl<cetostearyl. In niosomes containing PGDS, myristyl alcohol (MA), and polysorbate, the fatty acid chain length of polyoxyethylene sorbitan ester-type surfactants has influenced the release rate and encapsulation efficiency. Based on the release rate, polysorbates can be ranked as polysorbate-20 (C(12))<polysorbate-60 (C(18))<polysorbate-80 (C(9=9))<polysorbate-40 (C(16)). In niosome preparation containing polysorbate-20 and dioctyl sodium sulfosuccinate (anionic surfactant), the release rate was slower than niosomes containing polysorbate-20. When MA concentration is kept constant at 50 mole% and the ratio of PGDS and PS-80 was altered, significant changes in entrapment efficiency and the release rate were observed. However, this ratio did not exhibit any relation with encapsulation efficiency or release rate. The release rate and entrapment exhibited an inverse correlation (r(2)=0.8774 at p<0.02 for the data of molar ratios of PGDS:MA:PS80; r(2)=0.975 at p<0.001 for the data of acyl chain length variation of polysorbates). It can be concluded that stable niosomes of polyglyceryl-3-di-isostearate could be prepared with fatty alcohols and polysorbates instead of cholesterol and that the release of solutes from these niosomes can be optimized by altering membrane constituents and their concentrations.