Three cholesterol precursors-desmosterol, zymesterol, and lanosterol-were comprehensively characterized in monolayers formed at the air/water interface. The studies were based On registration of the surface pressure (pi)-area (A) isotherms complemented with in situ analysis performed with application of modern physicochemical techniques: grazing incidence X-ray diffraction (GDCD) and Brewster angle microscopy (BAM). In this approach we were interested in the correlation between molecular structures of the studied sterols found in the cholesterol biosynthetic pathway and their membrane properties. Out results revealed that only desmosterol behaves in Langmuir monolayers comparably to cholesterol, the molecules of which arrange in the monolayers into a hexagonal lattice, while the two remaining sterols possess extremely different properties. We found that molecules of both zymosterol and lanosterol are organized on the water surface in the two-dimensional oblique unit cells despite the fact that they are oriented perpendicular to the monolayer plane: The comparison of chemical structures of the investigated sterols leads to the conclusion that the only structural motive that can be responsible for such unusual behavior is the double bond in the B sterol ring, which is located in desmosterol in a different position from in the other two sterols. This issue, which was neglected in the scientific literature, seems to have crucial importance for sterol activity in biomembranes. We showed that this structural modification in sterol molecules is directly responsible for their adaptation to proper functioning in biomembranes.