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A highly specific and sensitive mass assay for inositol hexakisphosphate (InsP6) was characterized. This centres around phosphorylating InsP6with [32P]ATP using a recombinant InsP6kinase from Giardia lambia, followed by HPLC of the 32P-labelled products with an internal [3H]InsP7standard. This assay was used to quantify InsP6levels in a variety of biological samples. Concentrations of InsP6in rat tissues varied from 10–20 μM (assuming 64% of wet weight of tissue is cytosol water), whereas using the same assumption axenic Dictyostelium discoideum cells contained 352±11 μM InsP6. HeLa cells were seeded at low density and grown to confluence, at which point they contained InsP6levels per mg of protein similar to rat tissues. This amounted to 1.952±0.117 nmol InsP6per culture dish, despite the cells being grown in serum shown to contain no detectable (less than 20 pmol per dish) InsP6. These results demonstrate that mammalian cells synthesize all their own InsP6. Human blood was analysed, and although the white cell fraction contained InsP6at a concentration comparable with other tissues, in serum and platelet-free plasma no InsP6was detected (<1 nM InsP6). Human urine was also examined, and also contained no detectable (<5 nM) InsP6. These results suggest that dietary studies purporting to measure InsP6at micromolar concentrations in human plasma or urine may not have been quantifying this inositol phosphate. Therefore claims that administrating InsP6in the diet or applying it topically can produce health benefits by increasing extracellular InsP6levels may require reassessment. [ABSTRACT FROM AUTHOR]