Smith-Lemli-Opitz syndrome (SLOS) is a hereditary disorder in which a defective gene encoding 7-dehydrocholesterol reductase causes the accumulation of noncholesterol sterols, such as 7-and 8-dehydrocholesterol. Using rigorous analytical methods in conjunction with a large collection of authentic standards, we unequivocally identified numerous noncholesterol sterols in 6 normal and 17 SLOS blood samples. Plasma or erythrocytes were saponified under oxygen-free conditions, followed by multiple chromatographic separations. Individual sterols were identified and quantitated by high performance liquid chromatography (HPLC), Ag ؉ -HPLC, gas chromatography (GC), GC-mass spectrometry, and nuclear magnetic resonance. As a percentage of total sterol content, the major C 27 sterols observed in the SLOS blood samples were cholesterol (12-98%), 7-dehydrocholesterol (0.4-44%), 8-dehydrocholesterol (0.5-22%), and cholesta-5,7,9(11)-trien-3  -ol (0.02-5%), whereas the normal blood samples contained Ͻ 0.03% each of the three noncholesterol sterols. SLOS and normal blood contained similar amounts of lathosterol (0.05-0.6%) and cholestanol (0.1-0.4%) and ϳ 0.003-0.1% each of the ⌬ 8 , ⌬ 8(14) , ⌬ 5,8(14) , ⌬ 5,24 , ⌬ 6,8 , ⌬ 6,8(14) , and ⌬ 7,24 sterols. The results are consistent with the hypothesis that the ⌬ 8(14) sterol is an intermediate of cholesterol synthesis and indicate the existence of undescribed aberrant pathways that may explain the formation of the ⌬ 5,7,9(11) sterol. 19-Norcholesta-5,7,9-trien-3  -ol was absent in both SLOS and normal blood, although it was routinely observed as a GC artifact in fractions containing 8-dehydrocholesterol. The overall findings advance the understanding of SLOS and provide a methodological model for studying other metabolic disorders of cholesterol synthesis.-Ruan, B.