Serine proteases play a key function in extracellular processes affecting central nervous system plasticity. Recently, the role of extracellular proteolytic processes in regulating synaptic structure and function has been described. However, to date direct evidence linking extracellular serine protease activity with drug-related behavioural changes has not been documented. Importantly, in a screening for genes induced after drug treatment we found that urokinase plasminogen-type activator (uPA) was strongly regulated by cocaine in several protocols of drug administration. Cocaine-induced up-regulation could be verified on microarray analysis under several protocols of drug administration, then further fully confirmed by means of qRT-PCR. As a result, we chose to investigate further uPA function in the mesolimbic dopaminergic pathway, a major target area of cocaine and drugs of misuse. Our approach was based on the characterization of cocaine-induced behavioural changes following lentiviral vector delivery of a doxycycline-regulated uPA expression cassette (or of its mutated form), into specific rat brain areas (the hippocampus, the nucleus accumbens and the ventral tegmental area). We show that doxycycline-dependent over-expression of uPA in these regions yields a 10- to 12.3-fold increase in locomotor activity after cocaine administration. These behavioural effects were completely abolished when the active site of the protease was point-mutated and used as a dominant negative. The physiological relevance of these drastic behavioural changes is discussed.