Locomotor pattern generation is maintained by integration of the intrinsic properties of the spinal central pattern generator (CPG) neurons in conjunction with synaptic activity of the neural network. In the lamprey, the spinal locomotor CPG is modulated by 5-HT. On a cellular level, 5-HT presynaptically inhibits synaptic transmission and postsynaptically inhibits a Ca²⁺ -activated K⁺ current responsible for the slow afterhyperpolarization (AHP) that follows action potentials in ventral horn neurons. To understand the contribution of these cellular mechanisms to the modulation of the spinal central pattern generator, we have tested the effect of selective 5-HT analogues against fictive locomotion initiated by bath application of NMDA. We find that the 5-HT_1D agonist, L694-247, dramatically prolongs the frequency of ventral root bursting. Furthermore, we demonstrate that L694-247 presynaptically inhibits synaptic transmission without altering postsynaptic Ca²⁺ -activated K⁺ currents. We also confirm that 5-HT inhibits synaptic transmission at concentrations that modulate locomotion. To examine the mechanism by which selective presynaptic inhibition modulates the frequency of fictive locomotion we performed voltage and current clamp recordings of CPG neurons during locomotion. Our results show that 5-HT decreases glutamatergic synaptic drive within the locomotor CPG during fictive locomotion. Thus, we conclude that presynaptic inhibition of neurotransmitter release contributes to 5-HT-medaited modulation of locomotor activity.