The serotonin 1A (5-HT 1A ) receptor is an important member of the superfamily of seven transmembrane domain G-protein coupled receptors (GPCRs). We report here that guanine nucleotide sensitivity of agonist binding to hippocampal 5-HT 1A receptors is dependent on the concentration of Mg 2+ . Our results show that agonist binding to 5-HT 1A receptors is relatively insensitive to guanine nucleotides in the absence of Mg 2+ . In contrast to this, the specific antagonist binding is insensitive to guanine nucleotides, even in the presence of Mg 2+ . These results point out the requirement of an optimal concentration of Mg 2+ which could be used in assays toward determining guanine nucleotide sensitivity of ligand binding to GPCRs such as the 5-HT 1A receptor. Our results provide novel insight into the requirement and concentration dependence of Mg 2+ in relation to guanine nucleotide sensitivity for the 5-HT 1A receptor in particular, and GPCRs in general.