We have examined the fractional sterol concentration dependence of dehydroergosterol (DHE) fluorescence in DHE/cholesterol/dimyristoyl-L-α- phosphatidylcholine (DMPC), DHE/ergosterol/DMPC and DHE/cholesterol/dipalmitoyl-L-α phosphatidylcholine (DPPC) liquid- crystalline bilayers. Fluorescence intensity and lifetime exhibit local minima (dips) whenever the total sterol mole fraction, irrespective of the DHE content, is near the critical mole fractions predicted for sterols being regularly distributed in hexagonal superlattices. This result provides evidence that all three of these naturally occurring sterols (e.g., cholesterol, ergosterol, and DHE) can be regularly distributed in the membrane and that the bulky tetracyclic ring of the sterols is the cause of regular distribution. Moreover, at the critical sterol mole fractions, the steady-state anisotropy of DHE fluorescence and the calculated rotational relaxation times exhibit distinct peaks, suggesting that membrane free volume reaches a local minimum at critical sterol mole fractions. This, combined with the well-known sterol condensing effect on lipid acyl chains, provides a new understanding of how variations in membrane sterol content change membrane free volume. In addition to the fluorescence dips/peaks corresponding to hexagonal superlattices, we have observed intermediate fluorescence dips/peaks at concentrations predicted by the centered rectangular superlattice model. However, the 22.2 mol% dip for centered rectangular superlattices in DHE/ergosterol/DMPC mixtures becomes diminished after long incubation (4 weeks), whereas on the same time frame the 22.2 mol% dip in DHE/cholesterol/DMPC mixtures remains discernible, suggesting that although all three of these sterols can be regularly distributed, subtle differences in sterol structure cause changes in lateral sterol organization in the membrane.