Abstract The estimation of hexametaphosphate by the metachromatic reaction with toluidine blue has been standardized using substrates of known composition. The metachromatic intensity is sensitive to changes in acidity and temperature. It is inhibited by salts of univalent metals and ammonium. There is no simple relationship between the phosphate content and the metachromatic effect of various samples of hexametaphosphate. The sample that showed the deepest intensity had the highest molecular weight; the sample with the lowest molecular weight had also the lowest metachromasy. Pyrophosphate, tripolyphosphate, trimetaphosphate, and tetrametaphosphate do not interfere in the estimation of hexametaphosphate. Using the metachromatic method of assay it has been possible to show that aqueous solutions of hexametaphosphate are remarkably stable and that decomposition in the presence of chilled trichloroacetic acid is negligible. A simple method has been worked out for the decomposition of the insoluble Ba ++ salt of hexametaphosphate by contact with exchange resin. There is no diminution in the metachromasy of hexametaphosphate during Ba ++ precipitation and the subsequent removal of the Ba ++.