Tachykinins are present in lamprey spinal cord. The goal of the present study was to investigate whether an endogenous release of tachykinins contributes to activity of the spinal network generating locomotor activity. The locomotor network of the isolated lamprey spinal cord was activated by bath-applied N-methyl-D-aspartate (NMDA) and the efferent activity recorded from the ventral roots. When spantide II, a tachykinin receptor antagonist, was bath-applied after reaching a steady state burst frequency (> 2 hours), it significantly lowered the burst rate as compared to control pieces from the same animal. In addition, the time to reach the steady state burst frequency (> 2 hours) was lengthened in spantide II. These data indicate that an endogenous tachykinin release contributes to the ongoing activity of the locomotor network by modulating the glutamate/glycine neuronal network responsible for the locomotor pattern. We also explored the effects of a 10 minute exogenous application of substance P (1 µM), a tachykinin, and showed that its effect on the burst rate depended on the initial NMDA induced burst frequency. At low initial burst rates (around 0.5 Hz), tachykinins caused a marked further slowing to around 0.1 Hz, whereas at higher initial burst rates it instead caused an enhanced burst rate as previously reported, and in addition a slower modulation (0.1 Hz) of the amplitude of the motor activity. These effects occurred during an initial period of around 1 hour, while a modest long lasting increase of the burst rate remained after more than 2 hours.