Phosphatidylinositol-3,4,5-trisphosphate (PtdInsP(3)) regulates diverse cellular functions, including cell proliferation and apoptosis, and has roles in the progression of diabetes and cancer. However, little is known about its production. Here, we describe fluorescent indicators for PtdInsP(3) that allow a spatio-temporal examination of PtdInsP(3) production in single living cells. After ligand stimulation, PtdInsP(3) levels increased to a larger extent at the endomembranes (that is, the endoplasmic reticulum and the Golgi) than at the plasma membrane. This increase was found to originate from in situ production at the endomembranes, a process stimulated directly by receptor tyrosine kinases endocytosed from the plasma membrane to the endomembranes. The demonstration of PtdInsP(3) production through receptor endocytosis addresses a long-standing question about how signalling pathways downstream of PtdInsP(3) are activated at intracellular compartments remote from the plasma membrane.