Within the CNS, distinct neurons may rely on different processes to modulate cytosolic Ca2+ depending on the network developmental phase. In particular, in the immature spinal cord, synchronous electrical discharges are coupled with biochemical signals triggered by intracellular Ca2+ waves. Nevertheless, the presence of neuronal-specific Ca2+ elevations independent from synaptic activity within mammalian spinal networks has not yet been described. The present report is the first description of repetitive calcium events generated by discrete ventral spinal neurons maintained in organotypic culture during in vitro maturation stages crucial for network evolution. Ventral interneurons in one-third of slices displayed spontaneous intracellular calcium transients suppressed by calcium-free extracellular solution or by application of cobalt, and resistant to blockers of network activity like TTX, CNQX, APV, strychnine or bicuculline. Our data suggest a primary role for mitochondria in intracellular calcium oscillations, because CCCP, that selectively collapses the mitochondrial electrochemical gradient, eliminated the ability of these neurons to show activity-independent calcium oscillations. Likewise, CGP-37157, a blocker of mitochondrial Na+/Ca2+ exchanger, inhibited oscillations in the majority of neurons. We propose that spontaneous Ca2+ transients, dynamically regulated by mitochondria, occurred in a discrete cluster of interneurons possibly to guide the development of synaptic connections.