The requirement of membrane cholesterol in maintaining ligand binding activity of the hippocampal serotonin(1A) receptor has previously been demonstrated. In order to test the stringency of the requirement of cholesterol, we depleted cholesterol from native hippocampal membranes followed by replenishment with 7-dehydrocholesterol. The latter sterol is an immediate biosynthetic precursor of cholesterol differing only in a double bond at the 7th position in the sterol ring. Our results show, for the first time, that replenishment with 7-dehydrocholesterol does not restore ligand binding activity of the serotonin(1A) receptor, in spite of recovery of the overall membrane order. The requirement for restoration of ligand binding activity therefore is more stringent than the requirement for the recovery of overall membrane order. These novel results have potential implications in understanding the interaction of membrane lipids with this important neuronal receptor under pathogenic conditions such as the Smith-Lemli-Opitz syndrome. (C) 2007 Elsevier Inc. All rights reserved.