Development of motoneuron electrical properties and excitability was studied in spinal cord explants of rat embryos cultured for 1–3 weeks. The morphological organization of the spinal cord and synaptic inputs onto motoneurons were maintained in organ culture. The rate of differentiation of motoneuron resting potential and increase in membrane excitability was similar in vitro and in vivo, suggesting that these properties were regulated by cellular signals or extracellular differentiation-promoting factors that were preserved in culture. However, maturation of input resistance, action potential threshold and action potential maximum rate of rise was slower than in vivo. Culturing spinal cord explants with their dorsal root ganglia attached did not facilitate motoneuron differentiation. The role of newly formed synaptic pathways in regulating the changes in motoneuron electrical properties was studied in the presence of blockers of synaptic transmission. Motoneuron differentiation was delayed in spinal cords cultured in the presence of TTX, indicating that electrical activity influenced the time course of their development. However, blocking synaptic transmission with antagonists of glutamate, glycine, and GABAA receptors did not affect the rate of motoneuron differentiation, suggesting that maturation of motoneuron phenotype was independent of activation of these transmitter-gated channels. Incubating spinal cords in medium containing high-K+, which increased the frequency of spontaneous potentials, reversed the inhibitory effect of TTX. Similar to TTX action, motoneuron development was retarded when synaptic release was chronically blocked with either tetanus toxin or omega- conotoxin, a Ca2+ channel blocker. These findings suggested that electrical activity in spinal cord explants modulated motoneuron differentiation via Ca(2+)-dependent synaptic release of neurotransmitters or neurotrophic factors.