Cultured immature hippocampal neurons from embryonic 17-day-old rats were used to explore activity-dependent regulation of neuronal phenotype differentiation in the developing hippocampus. The calbindin-D28k phenotype of the pyramidal neurons appeared during the first 6 days in culture, and was expressed by 12% of the cells on day 6. Daily stimulation with 50 mm KCl during the first 5 days in vitro increased the number of calbindin-D28k-positive pyramidal neurons without affecting neuronal survival. This effect was prevented by buffering extracellular Ca2+. Omega-agatoxin-IVA-sensitive Q-type and nitrendipine-sensitive L-type voltage-gated Ca2+ channels (VGCCs) carried Ca2+ currents and Ca2+ influx in immature pyramidal neurons at somata level. Blockade of these channels inhibited calbindin-D28k phenotype induced by 50 mm KCl. Conversely, glutamate-activated Ca2+ channel antagonists did not affect the KCl-induced calbindin-D28k phenotype. Chronic blockade of Q- and/or L-type VGCCs downregulated the normal calbindin-D28k development of immature pyramidal neurons without affecting neuronal survival, the somatic area of pyramidal neurons or the number of GABAergic-positive (γ-aminobutyric acid) interneurons. However, at later developmental stages, Q-type VGCCs lost their ability to control Ca2+ influx at somata level, and both Q- and L-type VGCCs failed to regulate calbindin-D28k phenotype. These results suggest that Q-type channels, which have been predominantly associated with neurotransmitter release in adult brain, transiently act in synergy with L-type VGCCs to direct early neuronal differentiation of hippocampal pyramidal neurons before the establishment of their synaptic circuits.