Nicotine, a primary component of tobacco, is one of the most abused drugs worldwide. Mesolimbic dopaminergic neurons mediate the rewarding effects of abused drugs, including nicotine. We show that the tissue plasminogen activator (tPA) - plasmin system regulates nicotine-induced reward and dopamine release in the nucleus accumbens (NAc) by activating proteinase-activated receptor 1 (PAR(1)). Nicotine-induced conditioned place preference and dopamine release in the NAc are diminished in tPA knockout (tPA-/-) mice. The defect of nicotine-induced dopamine release in tPA-/- mice is reversed by microinjection of either exogenous tPA or plasmin into the NAc. Acute nicotine treatment increases tPA protein levels and promoted the release of tPA into the extracellular space. The expression of PAR(1) on dopaminergic neurons is evident and the activation of PAR(1) by plasmin is demonstrated by assaying GTP-gammaS binding. Finally, nicotine-induced conditioned place preference and dopamine release are diminished in PAR(1)-/- mice. These findings suggest that targeting the tPA-plasmin-PAR(1) system would provide new therapeutic approaches for the treatment of nicotine dependence.