To examine the possibility that glutamate may be widely used as the transmitter for cerebellar mossy fibres, population responses of granule cells following electrical stimulation of these fibres were recorded in rat cerebellar slices using a gap technique. Several different vermal lobules (II, V, VIb, VIII, IXc and X) whose main mossy fibre afferents originate in different nuclei, were compared. The mossy fibre response was remarkably similar in appearance in all lobules. With 1.2 mM Mg2+ in the perfusing solution, and with a low rate of stimulation (0.05 Hz), the N-methyl-D-aspartate (NMDA) antagonist, D-2-amino-5-phosphonovalerate (APV, 30 μM), had little or no effect but all components of the response could be inhibited by the broad spectrum excitatory amino acid antagonist, kynurenate (3 mM), or by the relatively selective non-NMDA antagonist, 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX, 10 μM). Slow APV-sensitive components emerged during high frequency stimulation (30 - 150 Hz) or, with a low stimulation rate, on removal of Mg2+ or addition of bicuculline (30 μM). The results suggest that an excitatory amino acid, presumably glutamate, is the major mossy fibre transmitter in the cerebellum and that it activates both NMDA and non-NMDA receptors on granule cells.