Fourteen phosphoserines and one phosphothreonine have been localized in a partial amino acid sequence of bovine milk osteopontin. Twelve of the phosphoserines are located in a Ser-X-Glu/Ser(P) sequence motif, suggesting that the phosphorylations are catalyzed by the mammary gland casein kinase. Two phosphoserines were found not to be located in a mammary gland casein kinase recognition sequence. Instead these two phosphoserines were located in the motif Ser-X-X-Glu which is a recognition sequence for casein kinase II. These data indicate that there might be more than one kinase active in the phosphorylation of osteopontin isolated from bovine milk. Furthermore, the serine in the cell-binding sequence Arg-Gly-Asp-Ser was shown not to be phosphorylated.