One of the most prominent roles of metabotropic glutamate receptors (mGluRs) in the CNS is to serve as presynaptic receptors that inhibit transmission at glutamatergic synapses. Previous reports suggest that the presynaptic effect of group II mGluRs at corticostriatal synapses can be inhibited by activators of protein kinase C (PKC). We now report that activation of PKC inhibits the ability of group II and group III mGluRs to regulate transmission at three major synapses in the hippocampal formation. Thus, this effect may be a widespread phenomenon that occurs at glutamatergic synapses throughout the CNS. We also report that this response is not limited to PKC-activating phorbol esters but that activation of A3 adenosine receptors induces a PKC-dependent inhibition of group III mGluR function at the Schaffer collateral-CA1 synapse. In addition to inhibiting mGluR modulation of excitatory synaptic transmission, we found that activation of PKC reduces inhibition of forskolin-stimulated cAMP accumulation by group II and group III mGluRs, suggesting that the effect of PKC on mGluR signaling is not specific to their effects on neurotransmitter release. This led us to test the hypothesis that PKC acts upstream from effector proteins regulated by mGluRs and acts at the level of the receptor or GTP-binding protein. Interestingly, we found that PKC inhibited mGluR-induced increases in [35S]-GTPgammaS binding in cortical synaptosomes. These data suggest that PKC-induced inhibition of mGluR signaling may be mediated by the inhibition of coupling of mGluRs to GTP-binding proteins.