Cholesteryl hemisuccinate (CHS) is one of the cholesterol-mimicking detergents not observed in nature. 
It is, however, widely used in protein crystallography, in biochemical studies of proteins, and in 
pharmacology. Here, we performed an extensive experimental and theoretical study on the behavior of 
CHS in lipid membranes rich in unsaturated phospholipids. We found that the deprotonated form of CHS 
(that is the predominant form under physiological conditions) does not mimic cholesterol very well. 
The protonated form of CHS does better in this regard, but also its ability to mimic the physical 
effects of cholesterol on lipid membranes is limited. Overall, although ordering and condensing 
effects characteristic to cholesterol are present in systems containing any form of CHS, their 
strength is appreciably weaker compared to cholesterol. Based on the considerable amount of experimental 
and atomistic simulation data, we conclude that these differences originate from the fact that the ester 
group of CHS does not anchor it in an optimal position at the water-membrane interface. The implications 
of these findings for considerations of protein-cholesterol interactions are briefly discussed.