Electrical stimulation of the superior colliculus in rats elicits not only orienting movements, as it does in other mammals, but also behaviours resembling such natural defensive responses as prolonged freezing, cringing, shying, and fast running and jumping. To investigate the location of the cells mediating these behaviours, the superior colliculus was systematically mapped with microinjections of sodium L-glutamate (50 mM, 200 nl), and the resultant behavioural changes as assessed in an open field were analysed for defence-like responses. The main regions that gave defensive behaviour were (i) rostromedial superior colliculus (all layers), and (ii) both medial and lateral parts of the caudal deep layers. Cells in these areas project into the ipsilateral descending pathway. However, the cells of origin of this pathway are also found in collicular regions, such as rostral intermediate gray and parts of far caudal colliculus, that did not give defensive movements in response to glutamate stimulation. It is unclear whether this is because only parts of the ipsilateral pathway mediate defensive behaviours, or because glutamate is a relatively inefficient stimulating agent for these systems. An unexpected feature of the results was that at a number of collicular sites the nature of the defensive response changed with successive (up to three) injections of glutamate, often appearing to become more intense. Whether the mechanism underlying this potentiation is related to the conditioning of natural defensive behaviour is unknown.