Caseins are highly phosphorylated milk proteins assembled in large colloidal structures termed micelles. In the milk of ruminants, alphas1-casein has been shown to be extensively phosphorylated. In this report we have determined the phosphorylation pattern of human alphas1-casein by a combination of matrix-assisted laser desorption mass spectrometry and amino acid sequence analysis. Three phosphorylation variants were identified. A nonphosphorylated form, a variant phosphorylated at Ser18 and a variant phosphorylated at Ser18 and Ser26. Both phosphorylation sites are located in the amino acid recognition sequence of the mammary gland casein kinase. Notably, no phosphorylations were observed in the conserved region covering residues Ser70-Glu78, which is extensively phosphorylated in the ruminant alphas1-caseins.