We have recently shown that phospholipase C-γ, (PLC-γ) is activated by tau, a neuronal cell-specific microtubule-associated protein, in the presence of arachidonic acid. We now report that non-neuronal tissues also contain a protein that can activate PLC-γ in the presence of arachidonic acid. Purification of this activator from bovine lung cytosol yielded several proteins with apparent molecular sizes of 70-130 kDa. They were identified as fragments derived from an unusually large protein (~700 kDa) named AHNAK, which comprises about 30 repeated motifs each 128 amino acids in length. Two AHNAK fragments containing one and four of the repeated motifs, respectively, were expressed as glutathione S-transferase fusion proteins. Both recombinant proteins activated PLC-γ1 at nanomolar concentrations in the presence of arachidonic acid, suggesting that an intact AHNAK molecule contains multiple sites for PLC-γ activation. The role of arachidonic acid was to promote a physical interaction between AHNAK and PLC-γ1, and the activation by AHNAK and arachidonic acid was mainly attributable to reduction in the enzyme's apparent K(m) toward the substrate phosphatidylinositol 4,5-bisphosphate. Our results suggest that arachidonic acid liberated by phospholipase A2 can act as an additional trigger for PLC-γ activation, constituting an alternative mechanism that is independent of tyrosine phosphorylation.