Calcium is an important nutrient for both young and elderly people which can, among others, stimulate healthy bone growth and prevent osteoporosis. Therefore, nutrition with sufficient calcium is of great importance. The highest calcium nutrient density for all foods can be found in bovine milk, which contains on average 1.2 g Ca / L. To increase the amount of bioavailable calcium, milk can be fortified by the addition of different calcium salts. In this research the bioavailability and exchangeability of calcium in unfortified and fortified skimmed milk was studied by in vitro methods using equilibrium dialysis and ultracentrifugation to separate different fractions in milk. Radioactive 45Ca was used to follow the exchange behaviour of calcium between the three different fractions in milk: casein micelles, serum proteins, and soluble calcium. For unfortified milk it was found that 34.9 ± 2.2% of the calcium was present in the soluble phase, 9.1 ± 0.6% was bound to serum proteins, and 56 ± 1.6% was present in the casein micelles, which confirmed the results in literature. In addition, with the investigation of the exchange behaviour of 45Ca, it was confirmed that ~40% of calcium in casein micelles is hardly exchanged.To investigate the influence of calcium fortification on the calcium equilibrium in milk, six different calcium salts were selected to be synthesized: calcium chloride, calcium carbonate, calciumgluconate, tri-calcium di-citrate, calcium lactate, and tri-calcium phosphate. Of these, calcium chloride, calcium carbonate, calcium gluconate, and tri-calcium di-citrate were successfully synthesized. Further experiments were performed with calcium chloride and calcium carbonate, intrinsically labelled with 45Ca. From fractionation with ultracentrifugation, it was concluded that addition of calcium chloride led to an increase of calcium in the soluble phase, which might indicate an increase in bioavailability. Fortification of milk with 45Ca labelled calciumcarbonate did not lead to an exchange of 45Ca with the calcium present in milk, indicating that calcium carbonate is unlikely to make calcium in milk more bioavailable. In conclusion, a better understanding of the solubility and exchangeability of Ca in milk and the influence of milk fortification with calcium chloride and calcium carbonate is obtained by using 45Ca as a tracer.