An assay to detect the on-target effects of mGlu2/3 receptor antagonists in vivo would be valuable in guiding dosing regimens for the exploration of biological effects of potential therapeutic import. Multiple approaches involving blockade of mGlu2/3 receptor agoinist-driven behavioral effects in mice and rats were investigated. Most of these methods failed to provide a useful method of detection of antagonists in vivo (e.g., locomotor activity). In contrast, the mGlu2/3 receptor agonist LY379268 produced dose-dependent increases in body temperature of mice. The hyperthermic effects of LY379268 was abolished in mGlu2 and in mGlu2/3 receptor null mice but not in mGlu3 null mice. Hyperthermia was not produced by an mGlu8 receptor agonist. Agonist-induced hyperthermia was prevented in a dose-dependent manner by structurally-distinct mGlu2/3 receptor antagonists. The blockade was stereo-specific. Moreover, this biological readout was responsive to both orthosteric and to negative allosteric modulators of mGlu2/3 receptors. Antagonism of agonist-induced hyperthermia predicted antidepressant-like efficacy in the mouse forced swim test. As with the hyperthermic response, the antidepressant-like effects of mGlu2/3 receptor antagonists were shown to be due to mGlu2 and not to mGlu3 or mGlu8 receptors through the use of receptor knock-out mice. The ability to rapidly assess on-target activity of mGlu2/3 receptor antagonists enables determination of parameters for setting efficacy doses in vivo. In turn, efficacy-related data in the preclinical laboratory can help to set expectations of therapeutic potential and dosing in humans.