Parallel electrophysiological and neurochemical studies of development are reported for mouse spinal cord cell cultures. The time course of electrical activity and the stage-dependent effects of tetrodotoxin on levels of the neuronal enzyme choline acetyltransferase were compared to establish the presence of spontaneous electrical activity at a time when tetrodotoxin adversely affects development. The extracellular patch electrode makes it possible to examine the ongoing electrical activity of the small cells present in young cultures. A rapid increase in spontaneous electrical activity during the first 2 weeks in culture was found to correlate closely with the onset of tetrodotoxin-induced depression of choline acetyltransferase activity, supporting the idea that ongoing electrical activity plays a role in neuronal development. The development of inhibitory synaptic activity occurs gradually throughout the period of culture, whereas excitatory synaptic activity and action potentials develop in unison, reaching maximal levels during the 2nd week in culture. For all cultures tested, ranging in age from 9 to 45 days old, acute bath application of gamma-aminobutyric acid (GABA) abolished spontaneous electrical activity. Glycine is relatively ineffective in abolishing spontaneous activity in young cultures which have few inhibitory postsynaptic potentials (IPSPs), but glycine becomes as effective as GABA at a later stage of development. This suggests rather different timetables of development for GABA and glycine receptors, with glycine receptors developing in parallel with IPSPs.