The neurotrophins (BDNF, NT-3, NT-4/5 and NGF) modulate survival, differentiation and synaptic plasticity of CNS neurons. Here we investigated the intracellular signalling cascades that mediate synaptic secretion of neurotrophins (NTs). BDNF-GFP and NT-3-GFP were expressed in hippocampal neurons, and time lapse video microscopy was used to explore the intracellular messengers involved in the secretion process. DsRed labeled PSD95 was coexpressed to enable investigation of synaptically localized neurotrophin vesicles. Depolarization induced (50 mM K+) secretion of neurotrophins was critically dependent on the influx of extracellular Ca2+. Inhibition of L-type calcium channels with nifedipine (50 mM) inhibited NT secretion. Depletion of internal Ca2+ stores by thapsigargin (10 mM) or inhibition of the CaM kinase II using KN-62 (10 mM) both led to a partial inhibition of NT secretion. Together, these data indicate that depolarization induced secretion of NTs is triggered by the influx of extracellular Ca2+, and that it is amplified by additional release of Ca2+ from internal stores and subsequent activation of CaMKII. Elevation of intracellular nitric oxide (NO, with 100 mM SNP) partially inhibited NT secretion, while exogenously applied BDNF increased intracellular NO, indicating a cellular crosstalk between BDNF and NO signalling in synaptic plasticity. (Supported by the DFG SFB 553)