<jats:sec><jats:title>BACKGROUND AND PURPOSE</jats:title><jats:p>The chemokine receptor CXCR3 is a GPCR found predominantly on activated T cells. CXCR3 is activated by three endogenous peptides; CXCL9, CXCL10 and CXCL11. Recently, a small‐molecule agonist, VUF10661, has been reported in the literature and synthesized in our laboratory. The aim of the present study was to provide a detailed pharmacological characterization of VUF10661 by comparing its effects with those of CXCL11.</jats:p></jats:sec><jats:sec><jats:title>EXPERIMENTAL APPROACH</jats:title><jats:p>Agonistic properties of VUF10661 were assessed in a chemotaxis assay with murine L1.2 cells transiently transfected with cDNA encoding the human CXCR3 receptor and in binding studies, with [<jats:sup>125</jats:sup>I]‐CXCL10 and [<jats:sup>125</jats:sup>I]‐CXCL11, on membrane preparations from HEK293 cells stably expressing CXCR3. [<jats:sup>35</jats:sup>S]‐GTPγS binding was used to determine its potency to induce CXCR3‐mediated G protein activation and BRET‐based assays to investigate its effects on intracellular cAMP levels and β‐arrestin recruitment.</jats:p></jats:sec><jats:sec><jats:title>KEY RESULTS</jats:title><jats:p>VUF10661 acted as a partial agonist in CXCR3‐mediated chemotaxis, bound to CXCR3 in an allosteric fashion in ligand binding assays and activated G<jats:sub>i</jats:sub> proteins with the same efficacy as CXCL11 in the [<jats:sup>35</jats:sup>S]‐GTPγS binding and cAMP assay, while it recruited more β‐arrestin1 and β‐arrestin2 to CXCR3 receptors than the chemokine.</jats:p></jats:sec><jats:sec><jats:title>CONCLUSIONS AND IMPLICATIONS</jats:title><jats:p>VUF10661, like CXCL11, activates both G protein‐dependent and ‐independent signalling via the CXCR3 receptor, but probably exerts its effects from an allosteric binding site that is different from that for CXCL11. It could stabilize different receptor and/or β‐arrestin conformations leading to differences in functional output. Such ligand‐biased signalling might offer interesting options for the therapeutic use of CXCR3 agonists.</jats:p></jats:sec><jats:sec><jats:title>LINKED ARTICLE</jats:title><jats:p>This article is commented on by O'Boyle, pp. 895–897 of this issue. To view this commentary visit <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="http://dx.doi.org/10.1111/j.1476-5381.2011.01759.x">http://dx.doi.org/10.1111/j.1476‐5381.2011.01759.x</jats:ext-link></jats:p></jats:sec>