The pH of optimum activity of alkaline phosphatase from cow's milk depended on the substrate, being 10·1 for p-nitrophenylphosphate, 8·6 for phosphoserine, 8·0 for phosvitin and 6·8 for casein. Individual casein components were dephosphorylated more rapidly than mixtures of αs- and β-caseins or of αs-, β- and κ-caseins and micellar casein. Mixtures of 2 components involving κ-casein were more readily dephosphorylated than αs- and β-casein mixtures. At pH 6·8, lactose, whey proteins and phosphate ions had an inhibitory effect. β-Lactoglobulin had an inhibitory effect only when the pH of the reaction was lower than the optimum pH value of the enzyme. Mg2+ and Zn2+ were not inhibitory. The optimum conditions for dephosphorylation of casein are described.