It has been suggested recently that presynaptic kainate receptors (KARs) are involved in short-term and long-term synaptic plasticity at hippocampal mossy fiber synapses. Using genetic deletion and pharmacology, we here assess the role of GLU_K5and GLU_K6in synaptic plasticity at hippocampal mossy fiber synapses. We found that the kainate-induced facilitation was completely abolished in theGLU_K6^-/-mice, whereas it was unaffected in theGLU_K5^-/-. Consistent with this finding, synaptic facilitation was reduced in theGLU_K6^-/-and was normal in theGLU_K5^-/-. In agreement with these results and ruling out any compensatory effects in the genetic deletion models, application of the GLU_K5-specific antagonistLY382884[(3S,4aR,6S,8aR)-6-(4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid] did not affect short-term and long-term synaptic plasticity at the hippocampal mossy fiber synapses. We therefore conclude that the facilitatory effects of kainate on mossy fiber synaptic transmission are mediated by GLU_K6-containing KARs.