AbstractWe have shown that endogenous activation of type 5 metabotropic glutamate (mGlu5) receptors supports the maintenance of a pluripotent, undifferentiated state in D3 mouse embryonic stem cells cultured in the presence of leukaemia inhibitory factor (LIF). Here, we examined the interaction between LIF and mGlu5 receptors using as a read‐out the immediate early gene, c‐Myc. The selective mGlu5 receptor antagonist, 2‐methyl‐6‐(phenylenthynyl)pyridine (MPEP; 1 μm), reduced the increase in c‐Myc protein levels induced by LIF by enhancing c‐Myc ubiquitination. A reduction in c‐Myc levels was also observed following small interfering RNA‐mediated mGlu5 receptor gene silencing. MPEP reduced glycogen synthase kinase‐3β phosphorylation on Ser9, but increased phosphorylation of the phosphatidylinositol‐3‐kinase (PI‐3‐K) substrate, AKT. In our hands, activated PI‐3‐K reduced the stability of c‐Myc, because (i) the PI‐3‐K inhibitor, LY294002, prevented the reduction in c‐Myc levels induced by MPEP; and (ii) over‐expression of AKT promoted c‐Myc ubiquitination. All effects of MPEP were mimicked by protein kinase C (PKC) inhibitors and reversed by the PKC activator, tetradecanoylphorbol‐13‐acetate. We conclude that endogenous activation of mGlu5 receptors sustains the increase in c‐Myc induced by LIF in embryonic stem cells by inhibiting both glycogen synthase kinase‐3β and PI‐3‐K, both effects resulting from the activation of PKC.