Many developing networks exhibit a transient period of spontaneous activity that is believed to be important developmentally. Here we investigate the initiation of spontaneous episodes of rhythmic activity in the embryonic chick spinal cord. These episodes recur regularly and are separated by quiescent intervals of many minutes. We examined the role of motoneurons and their intraspinal synaptic targets (R-interneurons) in the initiation of these episodes. During the latter part of the inter-episode interval, we recorded spontaneous, transient ventral root depolarizations that were accompanied by small, spatially diffuse fluorescent signals from interneurons retrogradely labeled with a calcium-sensitive dye. A transient often could be resolved at episode onset and was accompanied by an intense pre-episode (approximately 500 ms) motoneuronal discharge (particularly in adductor and sartorius) but not by interneuronal discharge monitored from the ventrolateral funiculus (VLF). An important role for this pre-episode motoneuron discharge was suggested by the finding that electrical stimulation of motor axons, sufficient to activate R-interneurons, could trigger episodes prematurely. This effect was mediated through activation of R-interneurons because it was prevented by pharmacological blockade of either the cholinergic motoneuronal inputs to R-interneurons or the GABAergic outputs from R-interneurons to other interneurons. Whole-cell recording from R-interneurons and imaging of calcium dye-labeled interneurons established that R-interneuron cell bodies were located dorsomedial to the lateral motor column (R-interneuron region). This region became active before other labeled interneurons when an episode was triggered by motor axon stimulation. At the beginning of a spontaneous episode, whole-cell recordings revealed that R-interneurons fired a high-frequency burst of spikes and optical recordings demonstrated that the R-interneuron region became active before other labeled interneurons. In the presence of cholinergic blockade, however, episode initiation slowed and the inter-episode interval lengthened. In addition, optical activity recorded from the R-interneuron region no longer led that of other labeled interneurons. Instead the initial activity occurred bilaterally in the region medial to the motor column and encompassing the central canal. These findings are consistent with the hypothesis that transient depolarizations and firing in motoneurons, originating from random fluctuations of interneuronal synaptic activity, activate R-interneurons, which then trigger the recruitment of the rest of the spinal interneuronal network. This unusual function for R-interneurons is likely to arise because the output of these interneurons is functionally excitatory during development.