Abstract A series of thirty two 6-hydroxylated steroids were synthesized by selective reduction of the 4–5 double bond, the 3-oxo group, and/or the 20-oxo group of 6α- and 6β-hydroxyDOC. The different reactions leading to the production of specific isomers are discussed. The gas chromatographic and spectrometric characteristics of the methoxime-trimethylsilyl (MO-TMS) or trimethylsilyl (TMS) derivatives of the isomers obtained are given. The gas chromatographic separation of the syn- and anti -isomers of the methoxime in position 3 was found to be characteristic of the configuration of the hydroxyl in position 6. The difference between methylene unit values of syn- and anti- isomers is much larger for the 6α-series than for the 6β-series. The mass spectral analysis showed that many ions are specific of the MO-TMS derivatives of steroids with 3,6-dihydroxy-4-ene or 3-oxo-6-hydroxy-4-ene structure. In the case of steroids with a satureated ring A no significant ions characteristic of the presence of a 6-trimethylsilyloxy substituent were found. This work provides previously unavailable reference data on 6-hydroxylated steroids which should facilitate the study of corticosteroid metabolism.