We have tested the ability of several specific agonists of glutamate metabotropic receptors (mGluRs) to depress synaptic transmission at mossy fiber synapses in the CA3 region of the guinea pig hippocampus. 1S,3R-1-amino-cyclopentyl-1,3-dicarboxylate (ACPD) reversibly inhibited monosynaptic mossy fiber field potentials, presumably by a presynaptic mechanism, with an EC50 of 2.0 +/- 0.4 microM (n = 3), suggesting the presence of mGluRs on mossy fiber synaptic terminals of the group 1 or 2 category. L-2-amino-4-phosphono butanoate (L-AP4) also inhibited responses with an EC50 of 1.1 +/- 0.2 microM suggesting that mGluRs of the group 3 (mGluR4, 6, 7 and 8) category of receptors are also present on mossy fiber terminals. Both (2S,1'S,2'S)-2-(2'-carboxycyclopropyl)glycine (L-CCG1) and (S)-4-carboxy-3-hydroxy phenylglycine (4C3HPG) were also efficacious at blocking mossy fiber transmission, with an EC50 of 1.1 +/- 0.1 microM (n = 4) and 4.8 +/- 0.6 microM (n = 3) respectively. The latter finding indicates the involvement of mGluRs belonging to the group 2 (mGluR2, 3) category of receptors. The effects of L-AP4 and L-CCG1 were both antagonized by (+)-alpha-methyl-4-carboxyphenylglycine [(+)MCPG]. MAP4, an antagonist of group 3 mGluRs in other systems, blocked the effect of L-AP4, but not the effect of L-CCG1, while MCCG, an antagonist of group 2 mGluRs in other systems, blocked the effect of L-CCG1, but not the effect of L-AP4. These pharmacological findings provide strong evidence for the coexistence of group 2 and 3 mGluRs on the terminals of mossy fibers in the guinea pig.