Brevetoxins (PbTx-1 to PbTx-10) are potent lipid soluble polyether neurotoxins produced by the marine dinoflagellate Karenia brevis. Brevetoxins bind to site 5 of the alpha-subunit of voltage-gated sodium channels (VGSCs) and augment Na(+) influx. In neocortical neurons brevetoxins elevate intracellular Ca(2+) and augment NMDA receptor signaling. In this study, we explored the effects of PbTx-2 on Pyk2 and Src activation in neocortical neurons. We found that both Pyk2 and Src were activated following PbTx-2 exposure. PbTx-2-induced Pyk2 Tyr402 phosphorylation was dependent on elevation of Ca(2+) influx through NMDA receptors. Moreover, Pyk2 Tyr402 phosphorylation was also found to require PKC activation inasmuch as RO-31-8425 and GF 109203x both attenuated the response. In contrast, PbTx-2-induced Src Tyr416 phosphorylation involved a Gq-coupled receptor inasmuch as U73122, a specific PLC inhibitor, abolished the response. This Gq-coupled receptor appears to be mGluR 5. The PKCdelta inhibitor rottlerin abolished PbTx-2-induced Src activation demonstrating that this isoform of PKC is involved in the activation of Src by PbTx-2. Considered together these data suggest that although activation of neuronal Pyk2 and Src result from PbTx-2 stimulation of VGSC, engagement of these two non-receptor tyrosine kinases involves distinct signaling pathways.