We report here the first exploration of the nature of the hydrophobic region of bilayer membranes formed from sterol-modified phospholipids [Huang, Z.; Szoka, F. C., Sterol-Modified Phospholipids: Cholesterol and Phospholipid Chimeras with Improved Biomembrane Properties. J. Am. Chem. Soc. 2008, 130 (46), 15702-15712] & [Ding, J.; Starling, A. P.; East, J. M.; Lee, A. G., Binding Sites for Cholesterol on Ca(2+)-ATPase Studied by Using a Cholesterol-Containing Phospholipid. Biochemistry 1994, 33 (16), 4974-4979]. Using H-2 NMR spectroscopy, we present our results for the phase behavior and acyl chain ordering of multilamellar vesicles (MLVs) of a sterol-modified phospholipid, 1-cholesterylhemisuccinoyl-2-palmitoyl (d(31))-sn-glycero-3-phosphocholine (hereafter referred to as CholPPC-d(31)). We compared our results with the conformational order induced by cholesterol at various concentrations in 1-palmitoyl,2-palmitoyl (d(31))-sn-glycero-3-phosphocholine (DPPC-d(31))/cholesterol membranes. On the basis of the existing literature [Foglia, F.; Barlow, D. J.; Szoka, F. C.; Huang, Z.; Rogers, S. E.; Lawrence, M. J., Structural Studies of the Monolayers and Bilayers Formed by a Novel Cholesterol-Phospholipid Chimera. Langmuir 2011, 27 (13), 8275-8281], we expected to find that the deuterated palmitoyl chain in CholPPC-d(31) membranes had an order parameter profile similar to the deuterated palmitoyl chain of sn-2 labeled DPPC-d(31) in MLVs of a mixture of DPPC-d(31) with 40 mol % unconstrained cholesterol. Our data indicate that the ordering ability of cholesterol in CholPPC is significantly reduced compared to free cholesterol in DPPC. This result emphasizes that cholesterol molecules must be free to move in the bilayers to reach their maximum ordering ability. In other words, when compared to unconstrained cholesterol, the constrained cholesterol moiety in CholPPC causes nonoptimal chain packing.