Casein micelles of different composition were synthesized in various ways and their sub-structure was investigated with the electron microscope by means of thin sections. Earlier studies of Schmidt et al. (1974) using the freeze-fracturing technique had shown no differences in the sub-structure of natural micelles and artificial micelles containing Ca and casein only. Contrary to these results our present studies showed that for the production of synthetic casein micelles with the same sub-structure as the natural ones it is necessary to add at least 2 other ions to the casein solution besides Ca 2+ : these are phosphate and citrate. The citrate ions play an important role in forming the material of the dark framework of the micelles visible in electron micrographs of unstained thin sections. This supports the hypothesis of Pyne & McGann (1960) that casein micelles contain a citrate apatite.In recent years many scientists have synthesized artificial casein micelles of different compositions in different ways. Earlier studies of Schmidt et al. (1974) using the freeze-fracturing technique found, by electron microscopy, no differences in the sub-structure of natural micelles and artificial micelles containing Ca and casein only. In the present study the sub-structure of various synthetic casein micelles was investigated by the electron microscopy of thin sections of unstained micelles. In our previous investigations the natural casein micelles showed, in electron micrographs of thin sections, a typical sub-structure: a dark network indicating the location of the main portions of colloidal Ca, phosphate and citrate (Plate 2 a). In stereoscopical pictures this network has the structural features of a gel-like framework. MATERIALS AND METHODSFor the preparation of the micelles, first-cycle casein (Waugh et al. 1962) was used in various concentrations. In the final mixture the casein concentrations lay between 2-0 and 3-2%. Ca 2+ were added as CaCl 2 solutions, phosphate ions as di-sodium hydrogen phosphate or potassium di-hydrogen phosphate solutions and citrate ions as tri-potassium citrate solutions. In all experiments the concentrations of these solutions were chosen in such a way that the final mixture contained Ca, phosphate and citrate in the same concentrations as in milk. This means in molar concentrations 30 mM-Ca, 22 mM-phosphate (without the ester phosphate of casein) and 10 mMcitrate. All solutions were adjusted to pH 6-7 before use. The warmed solutions were mixed by stirring in 50-ml beakers in a water-bath at 37 °C. The solutions were added dropwise to the beakers using pipettes or micropipettes. Ten minutes after the end of the mixing the pH was adjusted to pH 6-7 with NaOH. Afterwards the mixture was allowed to stand for 1-3 h. The micelles were then fixed by adding 2-5 %

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