5-HT-stimulated [³⁵S]GTPγS binding to rat hippocampal membranes was pharmacologically characterized. Signal/noise ratio or percent increase over basal was optimized with 100 μM GDP, 2 - 10 mM MgCl₂, and 150 - 200 mM NaCl. However, we preferred the standard condition (20 μM GDP, 5 mM MgCl₂, and 100 mM NaCl: Condition I) to the alternative one (100 μM GDP, 5 mM MgCl₂, and 150 mM NaCl: Condition II) because 1) absolute values of basal and 5-HT-sensitive bindings decreased with higher concentrations of GDP and NaCl; 2) EC₅₀ values determined under Condition II were 2 - 6 fold higher than those under Condition I; 3) some partial agonists had less intrinsic activities in the presence of higher concentrations of GDP; and 4) Inhibitory effects of WAY100635 were complete under Condition I, while incomplete under Condition II. Pharmacological profile of concentration-dependent stimulation by a series of 5-HT ligands and concentration-dependent inhibition of 5-HT-stimulated binding by several 5-HT-receptor antagonists clearly indicated that this response under Condition I was mediated solely through 5-HT_1A receptors. Although caution should be paid especially to the apparent intrinsic activities susceptible to the assay conditions, this method appears useful to investigate functional coupling between 5-HT_1A receptors and their coupled G proteins in native hippocampal membranes.