Recently a number of arthropod hormones, considered to be 20-hydroxy-derivatives of ecdysone have been isolated by us from a crayfish [1], the oak silkmoth [2], a pine tree [3] (crustecdysone), silkworms [4] (20-hydroxy-ecdysone) and silkworms [6, 5a] (ecdysterone). The structures of these compounds are clearly very similar and from the published spectroscopic and physical data, it is likely that they have identical tetracycles. However, it is not known whether they have identical or epimeric configurations at C-20 and C-22. We now report on a careful comparison of these compounds isolated in our various laboratories. The 20-hydroxy-ecdysones (crustecdysone) isolated from arthropods [1, 2] and a plant [3] are identical in many respects, but the small amounts of materials isolated from arthropods did not permit exhaustive comparisons. However, the large amount of material available from the plant has permitted its isolation as a pure crystalline solid (m. p. 241--242.5 °) and close comparison with the pure crystalline 20-hydroxy-ecdysone [4] (m. p. 238--240 °) obtained from silkworms. A mixture of crustecdysone from a plant [8] and a sample of 20-hydroxy-ecdysone had m.p. 239--241.5 °. The two compounds showed identical behaviour on TLC (Kieselgel H (Merck) -- solvent system chloroform-ethanol 3:2) giving spots of the same Rf and colour on spraying with the vanillin/ sulphuric acid reagent. The mass spectrum of crustecdysone displayed the same fragment ions as that of 20-hydroxy-ecdysone. The ultraviolet absorption of pure crustecdysone (λmax 242 mμ, ε 12,400 in methanol) is in good agreement with the values for 20-hydroxy-ecdysone [4], and ecdysterone [6a] (λmax 240 mμ, ε 13,600 in ethanol). 20-Hydroxy-ecdysone and ecdysterone were compared in TLC (Kieselgel F (Merck -- solvent system chloroformmethanol 95:5, five times developed) and were found to be identical both in Rf and colour reaction with the vanillin/ sulphuric acid reagent. A mixture of ecdysterone and 20-hydroxyecdysone showed no depression of the melting point. Both the mass spectra of ecdysterone and 20-hydroxy-ecdysone contain the same peaks, especially the characteristic ones at 363 and 345. In addition, the infrared spectrum of crustecdysone (KBr disc) appears virtually superimposable on those of 20 hydroxy-ecdysone and of ecdysterone. The identity of "β-ecdysone" and 20-hydroxy-ecdysone has already been demonstrated [6]. There seems little doubt that crustecdysone and ecdysterone are identical and the same as 20-hydroxyecdysone. The ORD curve of crustecdysone from the plant in dioxane shows a positive Cotton effect, amplitude St, indicating that the A/B ring stereochemistry of this compound and ecdysone are the same.