Activation of metabotropic glutamate receptors (mGluRs) causes membrane hyperpolarization in midbrain dopamine neurons. This hyperpolarization results from the opening of Ca(2+)-sensitive K(+) channels, which is mediated by the release of Ca(2+) from intracellular stores. Neurotransmitter-induced mobilization of Ca(2+) is generally ascribed to the action of inositol 1,4,5-triphosphate (IP(3)) in neurons. Here we show that the mGluR-mediated Ca(2+) mobilization in dopamine neurons is caused by two intracellular second messengers: IP(3) and cyclic ADP-ribose (cADPR). Focal activation of mGluRs, attained by synaptic release of glutamate or iontophoretic application of aspartate, induced a wave of Ca(2+) that spread over a distance of approximately 50 microm through dendrites and the soma. Simultaneous inhibition of both IP(3)- and cADPR-dependent pathways with heparin and 8-NH(2)-cADPR was required to block the mGluR-induced Ca(2+) release, indicating a redundancy in the signaling mechanism. Activation of ryanodine receptors was suggested to mediate the cADPR-dependent pathway, because ruthenium red, an antagonist of ryanodine receptors, inhibited the mGluR response only when the cADPR-dependent pathway was isolated by blocking the IP(3)-dependent pathway with heparin. Finally, the mGluR-mediated hyperpolarization was shown to induce a transient pause in the spontaneous firing of dopamine neurons. These results demonstrate that an excitatory neurotransmitter glutamate uses multiple intracellular pathways to exert an inhibitory control on the excitability of dopamine neurons.