Sx1TV2/16C is a mouse embryonic stem (ES) cell line in which one copy of the Sox1 gene, an early neuroectodermal marker, has been targeted with a neomycin (G418) selection cassette. Directed differentiation with retinoic acid and G418 selection results in an enriched neural stem cell population that can be further differentiated into neurons. After 6-7 days post plating (D6-7PP) most neurons readily fired tetrodotoxin (TTX)-sensitive action potentials arising from the activation of tetrodotoxin (TTX)-sensitive Na+ channels. Neurons approached their maximal cell capacitance after D6-7PP, however ion channel expression continued until at least D21PP. The percentage of cells receiving spontaneous synaptic currents increased with days in culture until 100% of cells received a synaptic input by D20PP. Spontaneous synaptic currents were reduced in amplitude and frequency by TTX, or upon exposure to a Ca2+-free, 2.5 mM Mg2+ physiological saline. Synaptic currents of rapid decay time constants (<20 ms) were reduced in amplitude with membrane depolarization and preferentially blocked by the nonNMDA glutamatergic receptor antagonists, CNQX or NBQX. Ca2+ levels within ES cell-derived neurons increased in response to glutamate receptor agonists L-glutamate, AMPA, N-methyl-D-aspartate (NMDA) and kainic acid (KA) and to acetylcholine, ATP and dopamine. NBQX displaced the concentration-Ca2+ response curve to AMPA but not to glutamate or KA. NMDA evoked a cationic membrane current which reversed at -11 mV and displayed a Mg2+-dependent outward rectification (block) at negative potentials. Glycine and GABA evoked Cl--selective currents which reversed at -70 and 78 mV, respectively. It was concluded that ES-derived neurons fire action potentials, receive excitatory and inhibitory synaptic inputs and respond to various neurotransmitters in a manner similar to primary central neurons.