The rodent superior colliculus mediates a wide range of physiological and behavioural responses to sudden stimuli, including desynchronisation of the cortical electroencephalogram (EEG). To investigate how this desynchronisation is produced, one of two powerful excitatory agents, sodium L-glutamate (200 nl, 10 nmol) or bicuculline methiodide (200 nl, 40 pmol), was injected into the dorsal midbrain of sleeping rats. Microinjections at sites widely distributed throughout all layers of the superior colliculus were able to desynchronise the cortical EEG. i) In the superficial layers, bicuculline was effective at more sites than glutamate, whereas the reverse was true for the deep layers. ii) At some sites EEG desynchronisation occurred together with the defensive or orienting movements that are obtained from collicular stimulation in awake animals. At other sites cortical arousal occurred without such movements. iii) Comparison with a previous study suggested that urethane selectively blocks cortical arousal to glutamate injections in the superficial and intermediate grey layers. This evidence suggests that multiple collicular output pathways can desynchronise the cortical EEG, perhaps reflecting multiple functions for EEG desynchronisation.