Activity-induced neurogenesis via Ca2+ entry may be important for establishing Hebbian neural network. However, it remains unclear whether intracellular Ca2+ mobilization is required and which subtypes of Ca2+ release channels expressed in Ca2+ store organelles are involved in the activity-dependent neurogenesis. Here, we demonstrated that the activity of intracellular Ca2+ signaling, expression of neuronal transcription factor NeuroD, and the rate of neurogenesis were significantly inhibited in neuronal cells derived from embryonic stem (ES) cells deficient in the Ca2+ release channel type 2 ryanodine receptors (RyR2(-/-)). In wild-type (RyR2(+/+)) but not in RyR2(-/-) ES cells, activation of L-type Ca2+ channels, GABA(A) receptors, or RyRs promoted neuronal differentiation, while inhibition of these channels/receptors had an opposite effect. Moreover, neuronal differentiation promoted by activation of GABA(A) receptors or L-type Ca2+ channels in RyR2(+/+) cells was prevented by RyR inhibitors. No significant difference was detected in the expression level of GABA(A) receptors and L-type channels between neuronal cells derived from two types of ES cells. Thus, activity-induced Ca2+ influx through L-type Ca2+ channels alone is not sufficient in promoting neurogenesis. Instead, an intimate cooperation of L-type Ca2+ channels with RyR2 is crucial for the activity-dependent neurogenesis induced by paracrine and/or autocrine GABA signaling. (c) 2007 Elsevier Ltd. All rights reserved.