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A mouse embryonic stem (ES) cell line containing an inducible transgene for the proneural gene Neurogl has been used to generate glutamatergic neurons at a high efficiency. The present study used in vitro electrophysiology to establish the timeline for acquiring a functional neuronal phenotype in Neurog1-induced cells exhibiting a neuronal morphology. TTX-sensitive action potentials could be evoked from over 80% of the cells after only 4.5 days in vitro (DIV). These cells uniformly showed rapidly adapting responses to current injection, firing one to three action potentials at the onset of the stimulus. In the absence of Neurog1, a limited number of ES cells adopted a neuronal morphology, but these cells displayed slow calcium depolarizations rather than sodium-based spikes. Voltage-gated [Na.sup.+], [K.sup.+], and [Ca.sup.2+] currents were present in nearly all induced cells as early as 4.5 DIV. The voltage-dependent properties of these currents changed little from 4 to 12 DIV with half-activation voltage varying by < 10 mV for any current type throughout the culture period. This study demonstrates that forced expression of proneural genes can induce ES cells to quickly acquire a functional neuronal phenotype with mature electrophysiological properties. Transient overexpression of Neurog1 may be used in neural repair strategies that require the rapid induction of functional neurons from pluripotent stem cells. neuronal differentiation; neurogenesis; electrophysiology; basic helix-loop-helix; voltage-gated; ion channel doi: 10.1152/ajpcell.00207.2010.