During embryonic development chick and mouse spinal cords are activated by highly rhythmic episodes of spontaneous bursting activity at very early stages, while motoneurons are still migrating and beginning to extend their axons to the base of the limb. While such spontaneous activity has been shown to be important in refining neural projections once axons have reached their targets, early pathfinding events have been thought to be activity independent. However, in-ovo pharmacological manipulation of the transmitter systems that drive such early activity has shown that early motor axon pathfinding events are highly dependent on the normal pattern of bursting activity. A modest decrease in episode frequency resulted in dorsal-ventral pathfinding errors by lumbar motoneurons, and in the downregulation of several molecules required to successfully execute this guidance decision. In contrast, increasing the episode frequency was without effect on dorsal-ventral pathfinding. However, it prevented the subsequent motoneuron pool specific fasciculation of axons and their targeting to appropriate muscles, resulting in marked segmental pathfinding errors. These observations emphasize the need to better evaluate how such early spontaneous electrical activity may influence the molecular and transcription factor pathways that have been shown to regulate the differentiation of motor and interneuron phenotypes and the formation of spinal cord circuits. The intracellular signaling pathways by which episode frequency affects motor axon pathfinding must now be elucidated and it will be important to more precisely characterize the patterns with which specific subsets of motor and inter-neurons are activated normally and under conditions that alter spinal circuit formation.