Conventional protein kinase C (PKC) isoforms are abundant neuronal signaling proteins with important roles in regulating synaptic plasticity and other neuronal processes. Here, we investigate the role of ionotropic and metabotropic glutamate receptor (iGluR and mGluR, respectively) activation on the generation of Ca²⁺ and diacylglycerol (DAG) signals and the subsequent activation of the neuron-specific PKCγ isoform in hippocampal neurons. By combining Ca²⁺ imaging with total internal reflection microscopy analysis of specific biosensors, we show that elevation of both Ca²⁺ and DAG is necessary for sustained translocation and activation of EGFP (enhanced green fluorescent protein)-PKCγ. Both DAG production and PKCγ translocation were localized processes, typically observed within discrete microdomains along the dendritic branches. Markedly, intermediate-strength NMDA receptor (NMDAR) activation or moderate electrical stimulation generated Ca²⁺ but no DAG signals, whereas mGluR activation generated DAG but no Ca²⁺ signals. Both receptors were needed for PKCγ activation. This suggests that a coincidence detection process exists between iGluRs and mGluRs that relies on a molecular coincidence detection process based on the corequirement of Ca²⁺ and DAG for PKCγ activation. Nevertheless, the requirement for costimulation with mGluRs could be overcome for maximal NMDAR stimulation through a direct production of DAG via activation of the Ca²⁺-sensitive PLCδ (phospholipase Cδ) isoform. In a second important exception, mGluRs were sufficient for PKCγ activation in neurons in which Ca²⁺ stores were loaded by previous electrical activity. Together, the dual activation requirement for PKCγ provides a plausible molecular interpretation for different synergistic contributions of mGluRs to long-term potentiation and other synaptic plasticity processes.