In vitro methods for developmental neurotoxicity (DNT) testing have the potential to reduce animal use and increase insight into cellular and molecular mechanisms underlying chemical-induced alterations in the development of functional neuronal networks. Mouse neural progenitor cells (mNPCs) differentiate into nervous system-specific cell types and have proven valuable to detect DNT using biochemical and morphological techniques. We therefore investigated a number of functional neuronal parameters in primary mNPCs to explore their applicability for neurophysiological in vitro DNT testing.Immunocytochemistry confirmed that mNPCs express neuronal, glial and progenitor markers at various differentiation durations (1, 7, 14 and 21 days). Since intracellular calcium ([Ca(2+)]i) plays an essential role in neuronal development and function, we measured stimulus-evoked changes in [Ca(2+)]i at these differentiation durations using the Ca(2+)-responsive dye Fura-2. Increases in [Ca(2+)]i (averages ranging from 65-226 nM) were evoked by depolarization, adenosine triphosphate, L-glutamic acid, acetylcholine and dopamine (up to 87%, 57%, 93%, 28% and 37% responding cells, respectively), and to a lesser extent by serotonin and gamma-aminobutyric acid (both up to 10% responding cells). Notably, the changes in percentage of responsive cells and their response amplitudes over time indicate changes in the expression and functionality of the respective neurotransmitter receptors and related calcium signaling pathways during in vitro differentiation. The development of functional intercellular signaling pathways was confirmed using multi-electrode arrays, demonstrating that mNPCs develop electrical activity within 1-2 weeks of differentiation (55% active wells at 14 days of differentiation; mean spike rate of 1.16 spikes/s/electrode).The combined data demonstrate that mNPCs develop functional neuronal characteristics in vitro, making it a promising model to study chemical-induced effects on the development of neuronal function.