Minimal calcium in fresh human milk occurs in the fat. Freshly-collected milk was partitionedinto fat and skim milk fractions, and the skim milk was partitioned into a colloidal fractioncontaining casein micelles, and a diffusible fraction. This fractionation was repeated after freezingand thawing three times over 14 days. Up to 51% of the total calcium was found in the fat layerof the milk after sequential freezing and thawing. We aimed to contribute to the understanding ofthe calcium interactions that cause this redistribution. Freezing and thawing disrupts the fat globulemembrane and releases fatty acids. We calculated that the amount of fatty acids generated by freezingand thawing of the milk would bind only a minor proportion of the calcium that was transferredto the fat layer. In addition, the calcium that appeared in the fat layer was not removed from thecolloidal fraction and therefore there was no evidence of disruption of the casein micelles. Thissuggests that the majority of the calcium that appears in the fat of human milk after freezing andthawing can be attributed to increases in the binding of calcium to the fat globule membranes.