The phosphoinositides have well-defined roles in the control of cellular functions, including cytoskeleton dynamics, membrane trafficking, and cell signaling. However, the interplay among the phosphoinositides and their diffusible derivatives that originate through phospholipase A₂ action (the lysophosphoinositides and glycerophosphoinositols) remains to be fully elucidated. Here we demonstrate that in PCCl₃ rat thyroid cells, the intracellular levels of glycerophosphoinositol are finely modulated by ATP and norepinephrine through the P2Y metabotropic and -adrenergic receptors, respectively. The enzyme involved here is phospholipase A₂ IV (PLA₂IV), which in these cells specifically hydrolyzes phosphatidylinositol, forming lysophosphatidylinositol, glycerophosphoinositol, and arachidonic acid. This receptor-mediated activation of PLA₂IV leads to stimulation of PCCl₃ cell growth. The involvement of a PLA₂IV-mediated pathway is demonstrated by inhibition of the increase in intracellular glycerophosphoinositol levels and cell proliferation by specific inhibitors, RNA interference, and overexpression of the dominant-negative PLA₂IV_1-522. Modulation of PCCl₃ cell growth is not seen with inhibitors of arachidonic acid metabolism. In conclusion, these data characterize glycerophosphoinositol as a mediator of the purinergic and adrenergic regulation of PCCl₃ cell proliferation, defining a novel regulatory cascade specifically involving this soluble phosphoinositide derivative and widening the involvement of the phosphoinositides in the regulation of cell function.--Mariggiò, S., Sebastià, J., Filippi, B. M., Iurisci, C., Volonté, C., Amadio, S., De Falco, V., Santoro, M., Corda, D. A Novel pathway of cell growth regulation mediated by a PLA₂-derived phosphoinositide metabolite.