We reported previously that BDNF induced glutamate release was dependent on intracellular Ca2+ but not extracellular Ca2+ in cerebellar neurons (Numakawa et al., 1999). It was revealed that the release was through a non-exocytotic pathway (Takei et al., 1998; Numakawa et al., 1999). In the present study, we monitored the dynamics of intracellular Ca2+ and Na+ in cerebellar neurons, and investigated the possibility of reverse transport of glutamate mediated by BDNF. As reported, BDNF increased the intracellular Ca2+ level. We found that the Ca2+ increase induced by BDNF was completely blocked by xestospongin C, an IP3 receptor antagonist, and U-73122, a PLC-γ inhibitor. Xestospongin C and U-73122 also blocked the BDNF-dependent glutamate release, suggesting that the BDNF-induced transient increase of Ca2+ through the activation of the PLC-γ/ IP3 pathway was essential for the glutamate release. We found that BDNF induced a Na+ influx. This was blocked by treatment with TTX. U-73122 and xestospongin C blocked the BDNF-induced Na+ influx, suggesting that the Na+influx required the BDNF-induced Ca2+ increase. Next, we examined the possibility that a co-transporter of Na+ and glutamate was involved in the BDNF-induced glutamate release. BDNF-induced glutamate release was blocked by L-trans-pyrollidine-2,4-dicalboxylic acid (t-PDC), a glutamate transporter inhibitor, whereas neither the 4-aminopyridine (4AP)- nor high potassium (HK+)-induced release was blocked by t-PDC. In addition, DL-threo-β-benzyloxyaspartate (DL-TBOA) also blocked the BDNF-mediated glutamate release, suggesting that reverse transport of glutamate may be involved. All the results therefore suggest that Na+-dependent reverse transport contributes to BDNF-mediated transmitter release through the PLC-γ/IP3-mediated Ca2+ signaling. J. Neurosci. Res. 66:96–108, 2001. © 2001 Wiley-Liss, Inc.