Background and Purpose The 5-HT receptor is a ligand-gated ion channel that is modulated allosterically by various compounds including colchicine, alcohols and volatile anaesthetics. However the positive allosteric modulators (PAMs) identified to date have low affinity, which hinders investigation because of non-selective effects at pharmacologically active concentrations. The present study identifies 5-chloroindole (Cl-indole) as a potent PAM of the 5-HT receptor. Experimental Approach 5-HT receptor function was assessed by the increase in intracellular calcium and single-cell electrophysiological recordings in HEK293 cells stably expressing the h5-HTA receptor and also the mouse native 5-HT receptor that increases neuronal contraction of bladder smooth muscle. Key Results Cl-indole (1-100 µM) potentiated agonist (5-HT) and particularly partial agonist [(S)-zacopride, DDP733, RR210, quipazine, dopamine, 2-methyl-5-HT, SR57227A, meta chlorophenyl biguanide] induced h5-HT A receptor-mediated responses. This effect of Cl-indole was also apparent at the mouse native 5-HT receptor. Radioligand-binding studies identified that Cl-indole induced a small (~twofold) increase in the apparent affinity of 5-HT for the h5-HTA receptor, whereas there was no effect upon the affinity of the antagonist, tropisetron. Cl-indole was able to reactivate desensitized 5-HT receptors. In contrast to its effect on the 5-HT receptor, Cl-indole did not alter human nicotinic a7 receptor responses. Conclusions and Implications The present study identifies Cl-indole as a relatively potent and selective PAM of the 5-HT receptor; such compounds will aid investigation of the molecular basis for allosteric modulation of the 5-HT receptor and may assist the discovery of novel therapeutic drugs targeting this receptor.