BACKGROUND AND PURPOSERecently identified antagonists of the urotensin-II (U-II) receptor (UT) are of limited utility for investigating the (patho)physiological role of U-II due to poor potency and limited selectivity and/or intrinsic activity. EXPERIMENTAL APPROACHThe pharmacological properties of two novel UT antagonists, GSK1440115 and GSK1562590, were compared using multiple bioassays. KEY RESULTSGSK1440115 (pKi = 7.34-8.64 across species) and GSK1562590 (pKi = 9.14-9.66 across species) are high affinity ligands of mammalian recombinant (mouse, rat, cat, monkey, human) and native (SJRH30 cells) UT. Both compounds exhibited >100-fold selectivity for UT versus 87 distinct mammalian GPCR, enzyme, ion channel and neurotransmitter uptake targets. GSK1440115 showed competitive antagonism at UT in arteries from all species tested (pA2 = 5.59-7.71). In contrast, GSK1562590 was an insurmountable UT antagonist in rat, cat and hUT transgenic mouse arteries (pKb = 8.93-10.12 across species), but a competitive antagonist in monkey arteries ). Likewise, GSK1562590 inhibited the hU-II-induced systemic pressor response in anaesthetized cats at a dose 10-fold lower than that of GSK1440115. The antagonistic effects of GSK1440115, but not GSK1562590, could be reversed by washout in rat isolated aorta. In ex vivo studies, GSK1562590 inhibited hU-II-induced contraction of rat aorta for at least 24 h following dosing. Dissociation of GSK1562590 binding was considerably slower at rat than monkey UT. CONCLUSIONS AND IMPLICATIONSWhereas both GSK1440115 and GSK1562590 represent high-affinity/selective UT antagonists suitable for assessing the (patho)physiological role of U-II, only GSK1562590 exhibited sustained UT residence time and improved preclinical efficacy in vivo.