Results from monolayer studies using the enantiomers of cholesterol (nat- and ent-cholesterol) provide the first evidence that enantioselective interactions between sterols and lipids can affect the physical properties of cell membranes. During monolayer compression, the ability of nat- and ent-cholesterol to act as enantioselective templates for ordering lipid alkyl chains into their extended conformations is found to depend on the structure of the lipid. Force/area isotherms for the compression of dipalmitoylphosphatidylcholine (L-DPPC) monolayers containing 10-60 mol% of either nat- or ent-cholesterol are not significantly different. Hence, there is no enantioselectivity for the ordering effect of cholesterol in these mixed films. By contrast, isotherms of egg yolk sphingomyelin (SPM) monolayers containing 20-40 mol% of either nat- or ent-cholesterol are clearly different indicating that enantioselective sterol-lipid interactions occur in these monolayers. Ent-cholesterol condenses egg yolk SPM monolayers more efficiently than does nat-cholesterol. At 30 mol%, where enantioselectivity is the greatest, the gaseous to liquid condensed phase transition starts at a mean molecular area of ~48 Å2/molecule for the ent-cholesterol/egg yolk SPM monolayer and at ~55 Å2/molecule for the nat-cholesterol/egg yolk SPM monolayer. Furthermore, Brewster angle microscopy shows that an additional phase is formed during compression of the 30 mol% ent-cholesterol/egg yolk SPM monolayers that is not formed during compression of the 30 mol% nat-cholesterol/egg yolk SPM monolayers. In a wider context, the results suggest that the physical properties of cholesterol-sphingomyelin rafts, cell membrane domains postulated to be important for the intracellular trafficking of proteins and as domains for plasma membrane proteins involved in cell-signaling pathways, are influenced by the absolute configuration of cholesterol.