The inferior olive climbing fibre projection is key to cerebellar contributions to motor control. Here we present evidence for a novel tool, trans-crotononitrile (TCN), to selectively inactivate the olive to study its functions. Anatomical, electrophysiological and behavioural techniques have been used in rats to assess the CNS effects of TCN, with a focus on the olivocerebellar projection. These findings were compared with those obtained with 3-acetylpyridine (plus nicotinamide administered 3.5 h later, 3AP + 3.5 h). Fluoro-Jade B cell labelling showed that TCN and 3AP + 3.5 h induce neurodegeneration primarily within the inferior olive, with no other targets in common. Recordings of evoked field potentials on the cerebellar cortical surface showed that both neurotoxins can reduce transmission in climbing fibre but not mossy fibre pathways. Both histological and electrophysiological differences suggest that TCN and 3AP have distinct mechanisms of action. Estimates of the numbers of surviving cells within individual subdivisions of the olive indicate that TCN and 3AP + 3.5 h cause different patterns of subtotal olivary lesion: most surviving neurons are present in the rostral (TCN) or caudal (3AP + 3.5 h) parts of the medial accessory olive, which are associated with two different cerebellar modules: the C2 and A modules, respectively. In behavioural studies, TCN and 3AP + 3.5 h produced differences in motor deficits consistent with the notion that these cerebellar modules have distinct functional responsibilities. Thus, studies using TCN as compared with 3AP + 3.5 h have the potential to shed light on the contributions of different cerebellar modules in motor control.