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The histochemical method employing potassium pyroantimonate in conjunction with electron microscopy has been investigated using carefully controlled preparation techniques and very sensitive atomic absorption analysis of cations. A critique on the reliability and limitations of the method based on test tube and in vitro experiments is given. The method is sensitive to Ca , Mg and Na+ at the 10-6, 10' and <102 M levels, respectively. Under defined conditions a linear '-.'l:l ratio of cation present to cation precipitated occurs above these levels. Approximate solubility products have been estimated. Under the test conditions, K' does not precipitate as a pyroantimonate salt, and neither K+ nor 0s04 influences cation precipitaton at physiologic concentrations. Unbuffered, Tris-HC1-buffered and weakly buffered NaHCO3 media at pH 7.2-7.8 give statistically similar results with Na+ precipitation. The pyroantimonate ion can compete with chelators, ethylenedinitrilotetraacetic acid and ethylene glycol bis-N,N'.tetraacetic acid, for divalent cations when employed simultaneously. These chelators effectively remove Ca '-but not Mg from embryonic myocardium, and their effects on Na+ and K+ balance are not marked if employed for relatively short periods. Electron micrographic examples of cation precipitates are given in support of certain findings. A brief discussion of the significance of pyroantimonate grain size, the discrepancy between the ratio of intra-and extracellular precipitates and guidelines for the use of the method are included.

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