To obtain greater insight into the interaction of Ca, citrate and phosphate ions with casein, 31PNMR measurements were performed on combinations of these ions with alphas- and kappa-caseins. It was found that addition of alphas-casein to a Ca phosphate solution in D2O at 27 degrees C and pD 6.4 resulted in a downfield shift of the 31P singlet. An almost identical shift was observed with kappa-casein, but no shift was found when only phosphate ions were present or when Ca2+ were added to phosphate ions in the absence of casein. Separate experiments with poly-L-lysine, mol. wt approx. 35,000, resulted in similar downfield 31P chemical shifts of Ca phosphate as with both caseins, whereas no shift was observed when poly-glycine was added. From these results it can be concluded that Ca and phosphate ions associate with casein in a co-operative manner, probably in the way described by ter Horst (1963) as a complex with the NH3+-groups of lysine or arginine in a structure such as: (casein--NH3+)--PO43---Ca2+. The formation of this complex may be enhanced by citrate ions, since preliminary results have shown that addition of Na citrate to a solution of alphas-casein with added Ca phosphate produces a broadening of the 31P signal as well as a chemical shift.