The primary objective of our study was to examine the role of the inhibitory neurotransmitters glycine and GABA in modulating spontaneous activity and coordinating neurochemically induced locomotor-like rhythms in the mouse spinal cord. Motor outputs were recorded in lumbar ventral roots of 1-4-day old neonatal mice, and the function of glycinergic and GABAergic synapses in regulating spontaneous and induced activities was examined by suppressing synaptic inhibition using selective glycine or GABAA receptor antagonists. Strychnine (0.5 microM), a glycine receptor antagonist, did not change the pattern of spontaneous activity that consisted of random single spikes and discharges of variable durations and intervals. In contrast, blocking GABAA receptors with either picrotoxin (10 microM) or bicuculline (5 microM) triggered bilaterally synchronous, non-rhythmic discharges. These findings suggested that GABAergic synapses suppressed excitatory synapses, and their disinhibition synchronized spontaneous discharges between the two sides of the spinal cord. Locomotor-like rhythms alternating between the two sides of the spinal cord were triggered by the neurotransmitter agonists 5-HT, N-methyl-D,L-aspartic acid and dopamine. Blocking glycine receptors increased tonic discharges, and in most preparations it reduced the phase correlation between the alternating rhythms. Inhibiting GABAA receptor-mediated synapses synchronized the onset and prolonged the duration of rhythmic discharges. Intraburst alternating peaks were evident and those were suppressed by strychnine, suggesting that they were mediated via glycinergic synapses. Our findings indicated that GABAergic and glycinergic synapses played different roles in modulating neurochemically induced locomotion rhythms. GABAergic inhibition regulated the onset and duration of neurochemically induced locomotor-like rhythms, and glycinergic inhibition stabilized the pattern of the alternating rhythms.