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Six new ecdysteroids have been isolated from Serratula tinctoria; these are: the 2,22- and 3,22-diacetates of 20-hydroxyecdysone, 5beta-hydroxyrubrosterone, 3-epi-poststerone, 3-epi-rubrosterone, and 22-oxo-20-hydroxyecdysone. These minor compounds were found together with the known ecdysteroids, 20-hydroxyecdysone, its 2-, 3-, and 22-monoacetates, rubrosterone, poststerone, polypodine B (5beta,20-dihydroxyecdysone), pterosterone (25-deoxy-20,24- dihvdroxyecdysone), and makisterone C (24-ethyl-20-- hydroxvecdysone). All these ecdysteroids were isolated by a combination of several chromatographic techniques (liquid chromatography on alumina, DCCC, and HPLC), then identified using standard mass spectrometric and 2D(1)H-NMR techniques.