Dihydropyrimidinase-like 3 (DPYSL3), a member of TUC (TOAD-64/Ulip/CRMP), is believed to play a role in neuronal differentiation, axonal outgrowth and possibly in neuronal regeneration. Recently, we have shown that in primary cortical neurons (PCN) NMDA and oxidative stress (H(2)O(2)) caused a calpain-dependent cleavage of DPYSL3 (62 kDa) resulting in the appearance of a lower molecular weight form (60 kDa) of DPYSL3. Our preliminary results had shown that antioxidants significantly reduced NMDA-induced DPYSL3 degradation, indicating involvement of ROS in calpain activation. The aim of this study was to investigate the possible involvement of NOS in NMDA-induced DPYSL3 degradation. We found that NOS inhibitor (L-NAME) significantly prevented NMDA-induced ROS formation, as well as intracellular Ca(2+) increase [Ca(2+)](i), DPYSL3 degradation and cell death. Further, exposure of PCN to NO donor (SNP) resulted in significant [Ca(2+)](i) increase, ROS generation and probable calpain-mediated DPYSL3 truncation. The NMDA- and oxidative stress (ROS)-induced DPYSL3 truncation was totally dependent on extracellular [Ca(2+)](i). While NMDA-induced DPYSL3 truncation was blocked by both NMDA receptor antagonist (MK801) [Kowara, R., Chen, Q., Milliken, M., Chakravarthy, B., 2005. Calpain-mediated degradation of dihydropyrimidinase-like 3 protein (DPYSL3) in response to NMDA and H(2)O(2) toxicity. J. Neurochem. 95 (2), 466-474] and L-VGCC (nimodipine) inhibitors, H(2)O(2)-induced increase in [Ca(2+)](i), ROS generation and DPYSL3 truncation was blocked only by nimodipine. These results indicate that changes in Ca(2+) homeostasis resulting from ROS-dependent activation of L-VGCC are sufficient to induce probable calpain-mediated DPYSL3 truncation and demonstrate for the first time the role of ROS in the mechanism leading to glutamate-induced calpain activation and DPYSL3 protein degradation. The probable calpain-mediated DPYSL3 truncation may have significant impact on its interaction with actin and its assembly, and in turn on growth cone integrity.