Cerebellar slices prepared from newborn rats were co-cultured with slices derived from the inferior olive of 4-day-old rats. After several weeks in vitro olivary fibres projecting into the cerebellar tissue could be assessed by anterograde labelling with the fluorescent dye 1,1-dioctadecyl-3,3,3,3′-tetramethylindocarbocyanine perchlorate (Dil). Following electrical field stimulation of the olivary tissue, all-or-nothing complex spikes were generated in Purkinje cells, which closely resembled climbing fibre responses as seen in situ. These responses were completely and reversibly abolished by 6-cyano-7-nitroquinoxaline-2-3-dione (CNQX, 5 μM), an antagonist of non-N-methyl-D-aspartate excitatory amino acid receptors. Wash in of smaller concentrations of CNQX (0.5–2 μM) resulted in a graded dose-dependent depression of the climbing fibre-induced postsynaptic potentials and in a consecutive failure of distinct active components of the complex spikes. With climbing fibre synaptic transmission blocked by CNQX, complex spike-like potentials could, however, still be evoked by intrasomatic injection of depolarizing current pulses. Increasing the concentration of Mg2+ in the bathing solution from 0.5 to up to 8 mM depressed regenerative complex-spike components. Olivary stimulation elicited only monophasic postsynaptic potentials in Purkinje cells under these conditions. These observations indicate that voltage-gated conductances which are substantially involved in the generation of the complex spike, are gated by the climbing fibre synaptic depolarization rather than directly by the climbing fibre transmitter.