There is increasing interest in the involvement of neurotrophins in neural transmission and plasticity. Thus, we investigated the effects of brain-derived neurotrophic factor (BDNF) on glutamate release from cortical neurons. Treatment of cultured cortical neurons with BDNF induced rapid and transient release of glutamate. This effect was suggested to be mediated by TrkB activation because K252a inhibited the release of glutamate and BDNF phosphorylated TrkB within 30 s. BDNF-induced glutamate release was observed even when using Ca2+-free assay buffer but was inhibited by BAPTA-AM, a cell-permeable Ca2+ chelator. Therefore, BDNF-induced glutamate release was independent of extracelluar Ca2+ but dependent on intracellular Ca2+. Because normal neurotransmitter release is exocytotic, the involvement of the exocytotic pathway in BDNF-induced glutamate release was examined. As botulinum toxin is known to cleave exocytosis-associated proteins, thereby inhibiting exocytosis, it was applied to neurons prior to the release assay. Although botulinum toxin B cleaved VAMP2 and inhibited Ca2+-triggered glutamate release, it did not inhibit the BDNF-induced release of glutamate. These results strongly suggested that BDNF induces rapid and transient release of glutamate from cortical neurons through a non-exocytotic pathway.