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Casein phosphopeptides (CPPs) form aggregated complexes with calcium phosphate and induce Ca2+ influx into HT-29 cells that have been shown to be differentiated in culture. The relationship between the aggregation of CPPs assessed by laser light scattering and their biological effect was studied using the CPPs β-CN(1–25)4P and αs1-CN(59–79)5P, the commercial mixture CPP DMV, the ‘cluster sequence’ pentapeptide, typical of CPPs, and dephosphorylated β-CN(1–25)4P, [β-CN(1–25)0P]. The biological effect was found to be: (a) maximal with β-CN(1–25)4P and null with the ‘cluster sequence’; (b) independent of the presence of inorganic phosphate; and (c) maximal at 4 mmol·L−1 Ca2+. The aggregation of CPP had the following features: (a) rapid occurrence; (b) maximal aggregation by β-CN(1–25)4P with aggregates of 60 nm hydrodynamic radius; (c) need for the concomitant presence of Ca2+ and CPP for optimal aggregation; (d) lower aggregation in Ca2+-free Krebs/Ringer/Hepes; (e) formation of bigger aggregates (150 nm radius) with β-CN(1–25)0P. With both β-CN(1–25)4P and CPP DMV, the maximum biological activity and degree of aggregation were reached at 4 mmol·L−1 Ca2+.