AbstractInositol phosphates control many central processes in eukaryotic cells, including nutrient availability, growth, and motility. Kinetic resolution of a key modulator of their signaling functions, the turnover of the phosphate groups on the inositol ring, has been hampered by slow uptake, high dilution, and constraining growth conditions in radioactive pulse-labeling approaches. Here, we demonstrate rapid (seconds to minutes), non-radioactive labeling of inositol polyphosphates through18O-water in yeast, amoeba and human cells, which can be applied in any media. In combination with capillary electrophoresis and mass spectrometry,18O-water labeling simultaneously dissects thein vivophosphate group dynamics of a broad spectrum of even rare inositol phosphates. The improved temporal resolution allowed us to discover vigorous phosphate group exchanges in some inositol poly- and pyrophosphates, whereas others remain remarkably inert. Our observations support a model in which the biosynthetic pathway of inositol poly- and pyrophosphates is organized in distinct, kinetically separated pools. While transfer of compounds between those pools is slow, each pool undergoes rapid internal phosphate cycling. This might enable the pools to perform distinct signaling functions while being metabolically connected.