Cocaine- and amphetamine-regulated transcript (CART) peptides have a wide CNS distribution and appear to play an important role in a number of physiological processes including reward and reinforcement, feeding, locomotion, stress responses and perception of pain. We have further investigated the expression of CART peptides in rat cerebellum, using multiple fluorescent antibodies. Dense fibre-like immunofluorescence was observed in the molecular layer of the vestibular cerebellum (paraflocculus and lobes IX and X of the vermis). There was little or no immunofluorescence in any other region of the cerebellum (vermis or hemispheres). The immunofluorescence in lobes IX and X of the vermis showed a parasagittal banding pattern, with one medial and two lateral bands. We have provided several lines of evidence that CART peptides are expressed by climbing fibres: (1) There is CART peptide immunofluorescence in the inferior olivary complex, the source of climbing fibres. (2) No cerebellar cell bodies were labelled by the CART peptide antibody. (3) The developmental profile of CART peptide expression was consistent with climbing fibre development and finally (4) triple antibody labelling revealed co-expression of CART peptides with VGluT2 (the glutamate transporter at climbing fibre synapses) at boutons innervating calbindin D-28K labelled proximal Purkinje cell dendrites. Analysis of VGluT2 and CART peptide labelling in more detail showed that not all climbing fibres in the vestibular cerebellum expressed CART peptides but those fibres which did express CART peptides were immunofluorescent at all Purkinje cell synapses.