The segmental organization of the hindlimb locomotor pattern generators and the coordination of rhythmic motor activity were studied in isolated spinal cords of the neonatal rat. All lumbar segments and many thoracic and sacral segments of the cord exhibited an alternating left-right rhythm in the presence of serotonin (5-HT) and N-methyl-D-aspartate (NDMA). Other thoracic segments exhibited a synchronized left-right rhythm or an irregular bursting activity. Transection of the cord at the thoracolumbar or lumbosacral junction abolished the rhythmicity of nonlumbar segments and had no affect on the rhythmicity of lumbar segments. A fast alternating rhythm persisted in rostral lumbar segments after transection of the cord at mid-L3. A much slower alternating rhythm was found in the detached caudal lumbar segments after elevation of the NMDA concentration. These findings suggest that neurogenesis of hindlimb locomotion is not restricted to L1/L2, and that the lumbar pattern generators exhibited rostrocaudal specialization. An alternating left-right rhythm persisted in lumbar cords of midsagittally split preparations that were kept with either L1, L2, L3, or L4 as the only bilaterally intact segment. An alternating rhythm persisted also in preparations that were midsagittally split up to T13-T12, or down to L4. Extension of these lesions led to a bilaterally synchronous rhythm or to left-right independent rhythms in the lumbar cord. These results indicated that the transverse coupling system in the caudal-thoracic and lumbar segments in specialized and that left-right alternation in the lumbar cord can be carried out by the cross connectivity, which is relayed at least through the T12-L4 segments. Bath application of the glycine receptor antagonist strychnine, or the gamma-aminobutyric acid-A (GABAA) receptor blocker bicuculline, induced in the presence of NMDA and 5-HT a bilaterally synchronous rhythm in any intact or detached segment of the cord and in midsagittally split preparations with few bilaterally intact upper thoracic or lower sacral segments. A strychnine-resistant left-right alternating rhythm was found in the presence of 5-HT and NMDA in preparations that were treated with the non-NMDA receptor blocker 6-cyano-7-nitroquinoxaline (CNQX) before and during the application of strychnine. Subsequent washout of CNQX immediately induced a bilateral synchronous rhythm. These results suggest that the phase relation between the hemicords during the rhythm is determined by a dynamic interplay between the excitatory and inhibitory cross connectivity, and that this interplay can be modulated experimentally. Local application of strychnine to L2 kept bilaterally intact in midsagittally split preparations perturbed but did not completely block the alternating pattern of the rhythm induced by 5-HT and NMDA. Local application of bicuculline under the same conditions prolonged the cycle time and had no effect on left-right alternation. These results, together with those described above, suggest that left-right alternation is mediated mainly by strychnine-sensitive glycine receptors with possible contribution of strychnine-resistant glycine receptors and/or GABAA receptors.