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The NK 3 receptor is a GPCR that is prominently expressed in limbic areas of the brain, many of which have been implicated in schizophrenia. Phase II clinical trials in schizophrenia with two selective NK 3 antagonists (osanetant and talnetant) have demonstrated significant improvement in positive symptoms. The objective of this study was to characterize the properties of a novel dual NK 2 /NK 3 antagonist, RO5328673. [ 3 H]RO5328673 bound to a single saturable site on hNK 2 , hNK 3 and gpNK 3 with high-affinity. RO5328673 acted as an insurmountable antagonist at both human and guinea-pig NK 3 receptors in the [ 3 H]IP accumulation assay. In binding kinetic analyses, [ 3 H]RO5328673 had fast association and dissociation rates at hNK 2 while it had a fast association rate and a remarkably slow dissociation rate at gp and hNK 3 . In electrophysiological recordings of gp SNpc, RO5328673 inhibited the senktide-induced potentiation of spontaneous activity of dopaminergic neurons with an insurmountable mechanism of action. RO5328673 exhibited in-vivo activity in gerbils, robustly reversing the senktide-induced locomotor activity. The TM2 residue gpNK 3 -A114 2.58 (threonine in all other species) was identified as the critical residue for the RO5328673's slower dissociation kinetics and stronger insurmountable mode of antagonism in the guinea-pig as compared to hNK 3 -T139 2.58 . Using site-directed mutagenesis, [ 3 H]RO5328673 binding and rhodopsin-based modeling, the important molecular determinants of the RO5328673-binding pocket of hNK 3 were determined. A comparison of the RO5328673-binding pocket with that of osanetant showed that two antagonists have similar contact sides on hNK 3 binding crevice except for three mutations V95L 1.42 , Y247W 5.38 , V255I 5.46 , which behaved differently between interacting modes of two antagonists in hNK 3 . [ABSTRACT FROM AUTHOR]