Networks generating locomotor-like rhythmic motor activity are formed during the last week of the fetal period in the rat spinal cord. We investigated the coordinated rhythmic motor activity induced in transverse slice preparations of the lumbar spinal cord taken from fetal rats as early as embryonic day (E) 16.5. In slices as thin as 100 µm, bath-application of 5-hydroxytryptamine (5-HT) induced rhythmic [Ca²⁺]_i elevations in motoneurons labeled with Calcium Green-1 dextran. The rhythmic [Ca²⁺]_i elevations were similar in frequency to that in the intact lumbar spinal cord, although there was no temporal correlation between the activity in the left and right sides of 100 µm slices. Such rhythmic [Ca²⁺]_i elevations were observed in the slices taken from all lumbar segments. Moreover, the rhythmic activity was abolished by simultaneous blockade of glutamate-, glycine- and GABA_A receptors, indicating that synaptic transmission mediated by these receptors is important for the generation of the rhythm in these slices. Synchronous rhythmic activity between the left-right sides was found in slices thicker than 200 µm taken from any segmental level of the lumbar spinal cord. In these preparations, commissural neurons were activated synchronously with ipsilateral motoneurons. These results indicate that the neuronal networks sufficient to generate coordinated rhythmic activity are contained in half of a single lumbar segment at E16.5. Such spinal cord slices are a promising experimental model to investigate the neuronal mechanisms and the development of rhythm generation in the spinal cord.