Absolute configuration at C-23 of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol, one of the bile alcohols isolated from the patients with cerebrotendinous xanthomatosis, was unequivocally determined as 23S by conversion of a key intermediate, (23S)-5 beta-cholest-25-ene-3 alpha,7 alpha,12 alpha,23-tetrol to either the bile alcohol of known absolute configuration, (23R)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,23-tetrol, or the naturally occurring 23,25-pentol.