It has been shown that peripherally administered substance P has reinforcing effects and can promote functional recovery after unilateral partial lesion of the nigrostriatal system. Furthermore, peripheral injection of substance P induces an increase in extracellular striatal dopamine. To obtain further information about the central mechanisms of these properties we used the in vivo microdialysis technique to investigate changes in the extracellular concentrations of acetylcholine in neostriatum and nucleus accumbens after intraperitoneal (i.p.) administration of substance P or vehicle in freely moving rats. The i.p. administration of 50 micrograms/kg substance P induced a steady, long-lasting decrease in the extracellular concentrations of acetylcholine in neostriatum, while no changes were observed in the nucleus accumbens. In comparison, substance P in a dose of 250 micrograms/kg i.p. acutely decreased the extracellular levels of acetylcholine in both nuclei. Interestingly, after the administration of vehicle, an acute increase in acetylcholine levels was observed in the nucleus accumbens, but not in the neostriatum. This effect did not occur after the injection of substance P indicating that the neurokinin blocked the increase in acetylcholine levels induced by the vehicle injection. These effects of substance P on striatal acetylcholine are discussed with respect to their relationship with dopamine and endogenous opiates, and with respect to the functional role of substance P, such as in reward, aversion, motor activity, and functional recovery.