The purpose of the study is to determine if expression or secretion of brain-derived neurotrophic factor (BDNF) in retinal ganglion cells (RGC-5) is mediated by NFkappaB or Ca2+/calmodulin-dependent protein kinase II (CaMKII). RGC-5 cells were exposed to 1 mM glutamate for various periods of time, in the presence or absence of prospective regulatory molecules. BDNF mRNA and protein expression were assessed with the aid of real-time PCR and immunoblots, respectively, and BDNF secretion was determined by ELISA. The NFkappaB inhibitor (TLCK and PTD-p65), or a specific CaMKII inhibitor (m-AIP), was used to study association of NFkappaB or CaMKII with BDNF expression/secretion in RGC-5 cells. Glutamate stimulated a transient increase in BDNF mRNA and protein in RGC-5 cells, and also stimulated an early release of BDNF into the culture media. Neutralizing the BDNF or blocking the TrkB receptor enhanced the glutamate-induced cytotoxicity. NFkappaB nuclear translocation was revealed in response to glutamate treatment. Application of TLCK or PTD-p65 inhibited the glutamate-induced BDNF expression and secretion. Inhibition of CaMKII by m-AIP did not affect expression but significantly enhanced the release of BDNF from glutamate challenged cells. Our data suggest that glutamate treatment may stimulate expression of BDNF in RGC-5 cells through NFkappaB activation. A novel mechanism for neuroprotection is proposed for the CaMKII inhibitor, AIP, which appears to protect RGC-5 cells from cytotoxicity by enhancing the release of BDNF from glutamate challenged cells.