AbstractRape seed oil methyl ester (RME), a diesel fuel substitute and a technical product of increasing importance, contains a number of minor components, which may influence its technical properties in various ways, Sterols, the main constituents of the unsaponifiable matter in rape seed oil, represent an important group of such minor components, which are then found in rape seed oil methyl ester. The concentration and composition of the total sterols in the fraction of unsaponifiables were determined in several RME samples of different origin by the usual procedure of saponification and isolation of the sterol fraction, followed by gas chromatographic analysis. The concentration and composition of the free sterols present in RME and the content of sterol esters were also determined by on‐line LC‐GC. The total sterol content in the RME samples varied between 0.70% and 0.81%. The sterol fraction of RME composed as follows: 0.4% cholesterol, 9.0% brassicasterol, 37.7% campesterol, 0.4% stigmasterol, 48.0% β‐sitosterol, 2.8% Δ5‐avenasterol, and 0.5% Δ7‐stigmasterol. By on‐line LC‐GC 0.24‐0.34% free sterols and 0.55‐0.71% sterol esters have been found in the RME samples.