Cholesterol, its precursor lanosterol, the plant sterols beta-sitosterol and ergosterol, as well as three further sterols were added up to 50 mol % to vesicles of the phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC). The aim of this study was to investigate the influence of the sterol side chain and ring structure on the acyl chain orientational order of the lipid bilayer by measuring the steady-state fluorescence anisotropy r(ss) of the fluorophore 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and to establish the molecular basis underlying the changes in order parameter of the lipid bilayer system. Experiments were carried out in the temperature range from 30 to 60 degreesC, i.e., at temperatures below and above the gel to liquid-crystalline phase transition temperature T-m of DPPC bilayers. In general, the incorporation of the different sterols into the DPPC bilayer enhances the orientational order of TMA-DPH in the liquid-crystalline state of DPPC, whereas the orientational order in the gel state is slightly reduced. We found that the molecular interactions between DPPC and sterols distinctly depend on the structure of the side chain and nucleus of the sterols. Compared to cholesterol, the presence of the additional ethyl group on the alkyl side chain of beta-sitosterol markedly reduces the effectiveness of the sterol on ordering the fluid DPPC bilayers over the whole concentration range of the sterol up to 50 mol %. As can be seen from the comparison with the other sterol mixtures, an increase in alkyl chain volume has the most drastic effect on the condensing capacity of sterols. Introduction of a double bond in the side chain counteracts this effect, owing to the decreased volume fluctuations in the steroid alkyl chain region. Interestingly, also introduction of a double bond in the sterol ring system leads at low sterol levels to a drastic increase in conformational order of the lipid acyl chains, which can even be substantially higher than that induced by cholesterol. Additional methyl groups in the ring system of the sterol markedly counteract this rigidifying effect. Hence, the lipids in the lanosterol-containing membrane are significantly less ordered than those in the cholesterol-containing membrane. Sterols with the bulkiest unsaturated side chains or sterol nuclei (stigmasterol, P-sitosterol, and lanosterol) induce the smallest order parameter increase of the fluid bilayer at high sterol concentrations (> 30 mol %), and hence become less potent rigidifiers at high sterol levels. At the highest sterol levels, cholesterol and the plant sterol ergosterol have the most profound ordering effect on fluid DPPC bilayers.