The spinal network underlying locomotion in lamprey is composed of excitatory and inhibitory interneurons mediating fast ionotropic action. In addition, several modulator systems are activated as locomotion is initiated, including the tachykinin system and the mGluR1 receptors, the latter operating partially via the endocannabinoid system. The effects of mGluR1 agonists and tachykinins resemble each other. Like mGluR1 agonists, the tachykinin substance P, accelerates the burst rate and reduces the crossed inhibition in an activity-dependent fashion. The present study therefore explores if tachykinins also utilize the endocannabinoid system to modulate the locomotor frequency. By monitoring fictive locomotion, we compare the facilitatory effects exerted by applying substance P (1 M, 20 min), on the burst frequency before and during application of the endocannabinoid CB1 receptor antagonist AM251 (2-5 M). It is shown that the response to substance P on the burst frequency is significantly reduced during the application of AM251 using two different lamprey species. To examine if endocannabinoids are involved in the substance P mediated modulation of reciprocal inhibition, the commissural axons were stimulated, while recording intracellularly from motoneurons. We compare the effect of substance P on the amplitude of the contralateral compound glycinergic IPSP in control and in the presence of AM251. The blockade of CB1 receptors reduced the substance P mediated decrease in the amplitude by 29%. The present findings suggest that the substance P effect on the increase in the locomotor burst frequency and depression of IPSPs are mediated partially via release of endocannabinoids acting through CB1 receptors.