The temperature-dependent dissociation of β-casein from the casein micelles of milk and from the soluble casein complexes of colloidal phosphate-free (CPF) milk was investigated by high-speed centrifugation and gel-filtration. The percentage of the total casein in supernatants prepared by high-speed centrifugation of mid-lactation milks increased from approximately 6 to 15% on cooling the milks from 30 to 5 °C; β-casein accounted for about 46% of this increase, while αs-and κ-casein constituted 30 and 23%, respectively. On gel-filtration both of skim-milk and CPF milk on Sepharose 2B at 0, 2, 5, 10 and 25 °C, maximum amounts of free β-casein (c. 60% of total) were obtained at 5 °C. The remainder of the β-casein appeared to be more strongly bound to the αs- and κ-casein and may be involved in the internal cohesion of casein micelles. The free β-casein of both milk preparations appeared to be in equilibrium with the bound β-casein. On Sephadex G-200 columns at 5 °C, approximately 5 and 60% of the β-casein of skim-milk and CPF milk, respectively, was eluted in the free form in the expected position for a globular protein of molecular weight about 200000. At low temperatures, particularly at 5 °C, colloidal phosphate appeared to play an integrating role in the association of over half the total β-casein with the other casein components of native micelles. However, when the equilibrium between micellar and free β-casein was disturbed by gel-filtration on Sepharose 2B, the presence of colloidal phosphate did not prevent the release of most of the β-casein from casein micelles. Some problems encountered in the use of densitometry for the estimation of individual caseins on electropherograms are described.