The lipid diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) was used to verify the existence of DAG-sensitive channels in cortical neurons dissociated from E13 mouse embryos. Calcium imaging experiments showed that OAG increased the cytosolic concentration of Ca2+ ([Ca2+]i) in nearly 35% of the KCl-responsive cells. These Ca2+ responses disappeared in a Ca2+-free medium supplemented with EGTA. Mn2+ quench experiments showed that OAG activated Ca2+-conducting channels that were also permeant to Ba2+. The OAG-induced Ca2+ responses were unaffected by nifedipine or omega-conotoxin GVIA (Sigma-Aldrich, Saint-Quentin Fallavier, France) but blocked by 1-[β-(3-(4-Methoxyphenyl)propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF)-96365 and Gd3+. Replacing Na+ ions with N-methyl-d-glucamine diminished the amplitude of the OAG-induced Ca2+ responses showing that the Ca2+ entry was mediated via Na+-dependent and Na+-independent mechanisms. Experiments carried out with the fluorescent Na+ indicator CoroNa Green showed that OAG elevated [Na+]i. Like OAG, the DAG lipase inhibitor RHC80267 increased [Ca2+]i but not the protein kinase C activator phorbol 12-myristate 13-acetate. Moreover, the OAG-induced Ca2+ responses were not regulated by protein kinase C activation or inhibition but they were augmented by flufenamic acid which increases currents through C-type transient receptor potential protein family (TRPC) 6 channels. In addition, application of hyperforin, a specific activator of TRPC6 channels, elevated [Ca2+]i. Whole-cell patch-clamp recordings showed that hyperforin activated non-selective cation channels. They were blocked by SKF-96365 but potentiated by flufenamic acid. Altogether, our data show the presence of hyperforin- and OAG-sensitive Ca2+-permeable channels displaying TRPC6-like properties. This is the first report revealing the existence of second messenger-operated channels in cortical neurons.