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An enzyme-linked immunosorbent assay (ELISA) of dehydroepiandrosterone sulfate (DHEAS), one of the neurosteroids, has been developed for measuring its brain and serum levels in rats without deconjugation. 11 alpha -Hemiglutaryloxy-DHEAS was newly synthesized, conjugated with bovine serum albumin (BSA), and immunized to rabbits for the production of anti-DHEAS antibodies. A bridge-heterologous ELISA system employing the sequential saturation method exhibited a high sensitivity with a midpoint of 100 pg. Although the antibody significantly cross-reacted with epiandrosterone sulfate, it easily discriminated the unconjugated steroids and pregnenolone sulfate, which is reported to exist in the brain at a much higher level when compared with DHEAS. The brain homogenate or serum was treated with hexane to remove the lipophilic compounds and purified with an OASIS HLB cartridge. The DHEAS levels were then determined by ELISA. The overall recovery rate through the pretreatment was a satisfactory and constant (81.8 +/- 3.4% for brain, 89.3 +/- 3.0% for serum, mean +/- standard deviation). This ELISA afforded a satisfactory serial dilution study and recovery test. The intra- and inter-assay coefficients of variation were lower than 13%, which showed the precision of the proposed method. The applied method showed that DHEAS was not detected in some brain samples and its levels were much lower than those previously reported and than its serum levels. (C) 2001 Elsevier Science Inc. All rights reserved.