The effects of cholesterol on various membrane proteins have received considerable attention. An important question regarding each of these effects is whether the cholesterol exerts its influence by binding directly to membrane proteins or by changing the properties of lipid bilayers. Recently it was suggested that a difference in the effects of natural cholesterol and its enantiomer, ent-cholesterol, would originate from direct binding of cholesterol to a target protein. This strategy rests on the fact that ent-cholesterol has appeared to have effects on lipid films similar to those of cholesterol, yet fluorescence microscopy studies of phospholipid monolayers have provided striking demonstrations of the enantiomer effects, showing opposite chirality of domain shapes for phospholipid enantiomer pairs. We observed the shapes of ordered domains in phospholipid monolayers containing either cholesterol or ent-cholesterol and found that the phospholipid chirality had a great effect on the domain chirality, whereas a minor (quantitative) effect of cholesterol chirality could be observed only in monolayers with racemic dipalmitoylphosphatidylcholine. The latter is likely to derive from cholesterol-cholesterol interactions. Accordingly, cholesterol chirality has only a modest effect that is highly likely to require the presence of solidlike domains and, accordingly, is unlikely to play a role in biological membranes.