Video-rate imaging of spinal neurons loaded with calcium-sensitive dyes was used to investigate the calcium dynamics and cellular organization of spontaneously active rhythm-generating networks in the spinal cord of E9-E12 chick embryos. Spinal neurons were loaded with bath-applied fura-2am. Motoneurons were also loaded by retrograde labeling with dextran-conjugated, calcium-sensitive dyes. Dye-filled motoneurons exhibited large fluorescent changes during antidromic stimulation of motor nerves, and an increase in the 340/380 fura fluorescence ratio that is indicative of increased intracellular free calcium. Rhythmic fluorescence changes in phase with motoneuron electrical activity were recorded from motoneurons and interneurons during episodes of evoked or spontaneous rhythmic motor activity. Fluorescent responses were present in the cytosol and in the perinuclear region, during antidromic stimulation and network-driven rhythmic activity. Optically active cells were mapped during rhythmic activity, revealing a widespread distribution in the transverse and horizontal planes of the spinal cord with the highest proportion in the ventrolateral part of the cord. Fluorescent signals were synchronized in different regions of the cord and were similar in time course in the lateral motor column and in the intermediate region. In the dorsal region the rhythm was less pronounced and the signal decayed after a large initial transient. Video-rate fluorescent measurements from individual cells confirmed that fluorescent signals were synchronized in interneurons and in motoneurons although the time course of the signal could vary between cells. Some of the interneurons exhibited tonic elevations of fluorescence for the duration of the episode whereas others were rhythmically active in phase with motoneurons. At the onset of each cycle of rhythmic activity the earliest fluorescent change occurred ventrolaterally, in and around the lateral motor column, from which it spread to the rest of the cord. The results suggest that neurons in the ventrolateral part of the spinal cord are important for rhythmogenesis and that axons traveling in the ventrolateral white matter may be involved in the rhythmic excitation of motoneurons and interneurons. The widespread synchrony of the rhythmic calcium transients may reflect the existence of extensive excitatory interconnections between spinal neurons. The network-driven calcium elevations in the cytosol and the perinuclear region may be important in mediating activity-dependent effects on the development of spinal neurons and networks.