1. The effects of metabotropic glutamate receptor (mGluR) agonists on excitatory postsynaptic potentials (EPSPs) evoked by stimulation of mossy fibers (MF) and parallel fibers (PF) were examined in turtle cerebellar Purkinje cells. 2. The mGluR agonist 1S,3R-ACPD (1-25 microM) reversibly potentiated the amplitude of the MF-evoked EPSPs, but was without effect on PF-evoked EPSPs. The potentiation of MF-evoked EPSPs was dose-dependent, with a median effective dose (ED50) of approximately 4.4 microM. At higher doses (15-25 microM) 1S,3R-ACPD produced a direct depolarization of Purkinje cells in 58% of cells examined. 3. The enhancement of MF EPSPs by 1S,3R-ACPD was mimicked by 1S,3S-ACPD (50 microM) and blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonovalerate (D-AP5), but not by the mGluR antagonist L-2-amino-3-phosphonopionic acid (L-AP3; 1 mM), or the 1R,3S isomer of ACPD (25-500 microM). 4. Quisqualate (1 microM) produced a biphasic effect on MF EPSPs, producing an initial blockade of the EPSP followed by a D-AP5-sensitive potentiation. 5. The potentiation of MF EPSPs by 1S,3R-ACPD was not blocked by prior exposure to the protein kinase C activator phorbol 12-myristate 13-acetate (10 microM), the protein kinase C inhibitor calphostin C (1 microM), the adenylate cyclase activator forskolin (25 microM), or the nitric oxide donator sodium nitroprusside (1 mM). Preincubation of the tissue for 24-48 h in pertussis toxin also failed to prevent the ability of 1S,3R-ACPD to potentiate the NMDA receptor-mediated component of the MF EPSP. PF EPSPs were also not significantly affected by these agents. 6. The results demonstrate that the mGluR agonists 1S,3R-ACPD, 1S,3S-ACPD, and quisqualate produce a potent, stereospecific potentiation of NMDA receptor-mediated transmission at the MF-granule cell synapse. Agents that modulate the intracellular messengers protein kinase C, adenylate cyclase, nitric oxide, or pertussis toxin-sensitive G proteins failed to mimic or block this effect. This would suggest that the potentiation of NMDA receptor-mediated transmission at this synapse is not mediated via these systems, and reflects a different site of action of mGluR agonists on the NMDA receptor. The observed interaction between mGluR and NMDA receptors in granule cells provides a means for activity-dependent modulation of synaptic transmission, which may play a role in synaptic integration at the MF-granule cell synapse.