Recent studies have demonstrated a functional interaction between group I metabotropic glutamate (mGlu) receptors and the cannabinoid system in the modulation of synaptic transmission. By using antisera directed against mGlu1alpha and CB1 cannabinoid receptors, we examined their distribution in the CA1 region of rat organotypic hippocampal slice cultures. Immunoreactive mGlu1alpha and CB1 elements were localized in non-principal cells, with a labeling distribution that was very similar to the pattern previously observed in the adult rat brain. Double-immunofluorescence staining and confocal microscopy showed that a subset of interneurons, mainly located in the stratum radiatum, was double-labeled for both mGlu1alpha and CB1 receptors. Co-localization of the two receptor subtypes was confirmed in hippocampal sections from adult rat brain. By using the "mirror technique" in adjacent sections, we observed that the double-labeled cells for mGlu1alpha and CB1 receptors were also immunopositive for the cholecystokinin peptide. Quantitative analysis revealed that in the stratum radiatum the majority (92%) of the CB1-positive cells and 19% of the mGlu1alpha-positive cells expressed both receptors. Triple immunofluorescence staining showed partial co-labeling of mGlu1alpha- and CB1-immunopositive cells with the vesicular glutamate transporter 3 or calbindin. Our results demonstrate that mGlu1alpha and CB1 receptors co-exist in a subpopulation of inhibitory neurons in the stratum radiatum of the hippocampus that is suggestive of the Schaffer collateral-associated interneurons. Hence, additional functional mechanisms underlying the cooperation between these two receptor subtypes may exist.