We quantified cytoplasmic Ca(2+) (Ca(2+)(c)) levels in cells dissociated from the embryonic (E) rat cortex during neurogenesis. Dual-recordings by flow cytometry using calcium and voltage-sensitive dyes revealed that, at the beginning of cortical development (E11-12), precursor cells exhibited either low (<100 nM), moderate (approximately 250 nM) or high (>1 microM) resting Ca(2+)(c) levels and well-polarized (-70 mV) or less-polarized (-40 mV) resting membrane potentials which reflected postmitotic or proliferative stages of the cell cycle. Ca(2+)(c) levels of all cells included a Ca(2+)(o) entry component, which was also Mn(2+)-permeant in actively proliferating precursors. Postmitotic, but not premitotic, precursors exhibited thapsigargin-sensitive intracellular Ca(2+) (Ca(2+)(i)) stores, which had similar capacities throughout neuronal lineage development. Differentiating neurons, but not precursors expressed Ca(2+)(i) stores with ryanodine and caffeine sensitivity and baseline Ca(2+)(c) levels that depended on Na(+)-Ca(2+) exchange activity. Voltage-dependent Ca(2+)(o) entry was not detected in precursors, but emerged during neuronal differentiation, with most of the neurons expressing functional L-type Ca(2+) channels. Ca(2+) imaging of individually immunoidentified cells acutely recovered in culture confirmed that precursors differentiate into neurons which stereotypically exhibit Ca(2+)(o) entry at the level of the membrane with increased Ca(2+)(i) release mechanisms on Ca(2+)(i) stores, Na(+)-Ca(2+) exchange activity and expression of voltage-dependent Ca(2+) channels.