Substance P initiates locomotion when injected in the brainstem of mammals. This study examined the possible role of this peptide on the supraspinal locomotor command system in lampreys. Substance P was bath applied or locally injected into an in vitro isolated brainstem and the effects of the drug were examined on reticulospinal cells and on the occurrence of swimming in a semi-intact preparation. Bath applications of substance P induced sustained depolarizations occurring rhythmically in intracellularly recorded reticulospinal cells. Spiking activity was superimposed on the depolarizations and swimming was induced. The sustained depolarizations were abolished by tetrodotoxin, and substance P did not affect the membrane resistance of reticulospinal cells, nor their firing properties, suggesting that it did not directly effect reticulospinal cells. To establish where the effects were exerted, successive lesions of the brainstem were made as well as local applications of the drug in the brainstem. Removing the mesencephalon abolished the sustained depolarizations, whereas large ejections of the drug in the mesencephalon excited reticulospinal cells and elicited bouts of swimming. More local injections into the mesencephalic locomotor region (MLR) also elicited swimming. After an injection of substance P, the current threshold needed to induce locomotion by MLR stimulation was decreased and the size of the postsynaptic responses of reticulospinal cells to MLR stimulation was increased. Substance P also reduced the frequency of miniature spontaneous postsynaptic currents in reticulospinal cells. Taken together, these results suggest that substance P plays a neuromodulatory role on the brainstem locomotor networks of lampreys.