Activity-dependent synaptic plasticity has been proposed as a contributory factor in the patterning of rhythmic network activity. However, its role has not been examined in detail. Here, paired or triple intracellular recordings have been made from identified neurons in the lamprey locomotor network to examine the potential relevance of activity-dependent synaptic plasticity in segmental and intersegmental spinal networks. Segmental inputs from glutamatergic excitatory interneurons (EIN) to ipsilateral glycinergic crossed caudal (CC) interneurons were facilitated or depressed during spike trains at 5-20 Hz. Connections between EINs were depressed. Glycinergic inputs from small ipsilateral inhibitory interneurons were depressed in motor neurons, but were facilitated in CC interneurons. Excitatory inputs from small crossing interneurons to motor neurons were depressed, whereas inhibitory inputs were unaffected. With the exception of connections between EINs, significant effects occurred with stimulation that mimicked interneuron spiking during network activity. Intersegmental EIN synaptic properties were also investigated. EIN inputs did not differ significantly when examined from zero to four segments rostral to motor neurons or CC interneurons. However, caudally located EINs evoked greater activity-dependent facilitation than did rostral EINs. Whilst the amplitude or plasticity of EIN inputs in the rostral or mid-trunk regions of the spinal cord did not differ, EINs in the caudal trunk region evoked greater facilitation. Synaptic transmission between locomotor network neurons thus exhibits activity-dependent plasticity in response to physiologically relevant stimulation. Activity-dependent plasticity could thus contribute to the patterning of the rhythmic network output.