Using an in vivo method for the assessment of polyphosphoinositide (PI) hydrolysis, we examine whether spatial learning and memory extinction cause changes in mGlu5 metabotropic glutamate receptor signaling in the hippocampus and prefrontal cortex. We use the following five groups of mice: (i) naive mice; (ii) control mice exposed to the same environment as learner mice; (iii) leaner mice, trained for four days in a water maze; (iv) mice in which memory extinction was induced by six trials without the platform; (v) mice that spontaneously lost memory. The mGlu5 receptor-mediated PI hydrolysis was significantly reduced in the dorsal hippocampus of learner mice as compared to naive and control mice. The mGlu5 receptor signaling was also reduced in the ventral hippocampus and prefrontal cortex of learner mice, but only with respect to naive mice. Memory extinction was associated with a large up-regulation of mGlu5 receptor-mediated PI hydrolysis in the three brain regions and with increases in mGlu5 receptor and phospholipase-Cβ protein levels in the ventral and dorsal hippocampus, respectively. These findings support a role for mGlu5 receptors in mechanisms underlying spatial learning and suggest that mGlu5 receptors are candidate drug targets for disorders in which cognitive functions are impaired or aversive memories are inappropriately retained.