It is known that only one stereoisomeric form, nat-cholesterol, naturally occurs. Nat-cholesterol and its enantiomer, ent-cholesterol, sometimes show enantiospecific interactions with biological molecules. If cholesterol is naturally found only one form, then the question of "why does cholesterol show an enantiomeric selectivity?" arises. For this purpose, stereoisomer analysis of cholesterol obtained from porcine liver and wool wax were carried out with three different high performance liquid chromatography (HPLC) systems including reversed-phase, reversed-phase with different cyclodextrins as a mobile phase modifier, and chiral. Results from HPLC analysis of both cholesterol samples by permethylated gamma-cyclodextrin and amylose tris-(3,5-dimethylphenylcarbamate) chiral columns showed that there was no stereoisomer of cholesterol present. However reversed-phase HPLC analysis of cholesterol samples from porcine liver carried out with various cyclodextrins as mobile phase modifiers presented a peak which was not observed in the analysis of cholesterol samples from wool wax. On the other hand, different storage conditions of cholesterol samples and addition of cyclodextrins as mobile phase modifiers produced almost identical alterations in chromatograms of fresh samples by reversed-phase HPLC. This could be attributed to catalytic properties of cyclodextrins. Cyclodextrins may not be suitable as a mobile phase modifier in the stereoisomer analysis of cholesterol with high performance liquid chromatography.