Cholesteryl nonanoate is monoclinic, space group P2₁, with lattice parameters a= 27.24(1), b= 9.183(3), c= 13.96(2)Å, β= 91.52(2)°, and Z= 4 [two independent molecules (A) and (B) in the unit cell]. The crystal structure was determined by Patterson rotation and translation methods, from the X-ray intensities of 2 496 reflections measured by diffractometer and refined by block-diagonal least-squares to R 0.10. Molecules (A) and (B) have almost fully extended conformations, but differ at the ends of the C(17) chains, in the rotations at the ester bonds, and in the nonanoate chains. The molecules are in antiparallel array forming monolayers with thickness d₁₀₀ 27.2 Å, and having molecular long axes tilted at ca. 61° with respect to the layer interface. In the interface region, atoms are almost in the liquid state. The crystal structure is unusual in that the nonanoate chains pack with cholesteryl tetracyclic systems and not with each other. Arrangements of this kind are presumed to exist when cholesterol is incorporated within biological membranes.