There is disagreement in the literature to whether or not there is a direct functional projection from the sensorimotor cerebral cortex to the inferior olive. A combination of electrophysiological and anatomical tract tracing techniques have been used in the present study to investigate the connectivity of this pathway. In pentobarbitone (50 mg/kg, i.p., Sagatal, Rhone Merieux, Harlow, UK) anaesthetised rats, single pulse electrical stimulation of the primary motor cortex evoked large climbing fibre potentials on the surface of the cerebellum. These inputs correspond with those defined by climbing fibre input from the periphery and were recorded in the C1 zone. Animals were killed humanely at the termination of the experiment. In anatomical studies, injections of anterograde tracer (Fluoro-Emerald and/or Fluoro-Ruby, Molecular Probes) were injected into the physiologically defined fore- and/or hindlimb areas of the motor cortex and retrograde tracerr (green beads and/or red beads, Lumafluor Inc.) into the C1 zone in the cerebellar cortex in the same animals. Overlaps between anterograde terminal and retrograde cell labelling were in the basal pontine nuclei. In contrast, only retrograde cell labelling was found in the inferior olive. Anterogradely labelled fibres continued past the inferior olive and after the decussation continued down to the spinal cord, although there was terminal labelling ventral to the gracile nucleus. In electrophysiological experiments, reversible inactivation of this anatomically identified area in the caudal brainstem with lignocaine resulted in a temporary abolition of climbing fibre field potentials evoked from cerebral cortical stimulation whilst responses evoked at the same C1 zone cerebellar cortical site by peripheral stimulation were unaffected. This suggests there is at least one relay prior to the inferior olive in the motor cortical cerebro-cerebellar pathway in rats.