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Dehydroepiandrosterone (DHEA), the native clinical steroid and steroid precursor, may have a targeted physiologic role. A high affinity (Kd 2.3nM) and steroid specific [3H] DHEA binding macromolecule in male Sprague-Dawley rat hepatic cytosol suggests that DHEA may have receptor mediated physiologic action. 3[H] DHEA binding was highest in the liver followed by kidney and testis cytosols. Sulfhydryl reagents such as N-ethylmalemide and iodoacetamide inhibited the binding of [3H]DHEA by up to 60-70%. The DHEA-macromolecular complex was stable at 35 degrees C. and addition of 5mM molybdate or 0.3M KCl increased stability. Interestingly, rat liver cytosol, specific binding at 4 degrees C increased by almost 40-50% with addition of 0.1M NaSCN or 0.3M KCl. Sucrose gradient analyses showed a 7-8 S macromolecular complex in the low salt and 3-4 S complex under high salt conditions. The [3H] DHEA- macromolecular complex shows minimal temperature dependent activation in vitro at 25 degrees C as judged by binding to DNA-cellulose. The results suggest a specific high affinity macromolecule for DHEA in rat liver cytosol with unique physicochemical properties.