We have investigated the spectroscopic evidence for sterol superlattice formation in bilayers composed of dehydroergosterol (DHE) and a homologous series of phosphatidylcholine, namely, C(18:0):C(18:1Delta")PC with n = 6, 7, 9, 11, and 13, These phosphatidyl;cholines (PCs) possess the same number of carbons and the same number of cis double bonds; however, the position of the double bond of these PCs varies. We have examined the fractional concentration dependence of the steady-state fluorescence intensity of DHE in the liquid-crystalline multilamellar vesicles composed of DHE and this series of PCs. The DHE contents in the membranes were varied by similar to0.2 mol % in two concentration regions, 18.7-21.3 and 23.9-26.0 mol % DHE. In these two regions, 20.0 and 25.0 mot % are the critical sterol mole fractions predicted for maximal superlattice formation. A DHE intensity dip appears at each of these two critical sterol mole fractions in the PC bilayers that have the double bond located between C9 and the terminal carbon. However, no distinct DHE intensity dips are observed in these two concentration regions as well as in the region 18.4-27.5 mot % DHE, when the double bond is located at C6-C7 or C7-C8. These data suggest that sterol superlattice can occur in liquid-crystalline state of unsaturated phospholipid bilayers so long as the cis double bonds are not located between C9 and the carboxyl carbon. It appears that appropriate van der Waals contacts between the steroid ring and the C2-C9 segment of the phospholipid acyl chain are required for sterol superlattice formation.