β-Casein was isolated from Haflinger mare's milk by RP-HPLC, and displayed microheterogeneity by urea-electrophoresis and 2-DE probably due to a variable degree of phosphorylation. To investigate the degree of phosphorylation, the primary structure of equine β-casein was determined by tryptic hydrolysis and MS of peptides released and by MS of the protein treated by alkaline phosphatase. The molecular mass found for the apo-form of Haflinger mare's β-casein (25 514 ± 3 Da) was close to the theoretical mass of the reported sequence (GenBank AAG43954) modified by insertion of a region (residues 27–34) encoded by an exon sometimes out-spliced (25 511.40 Da). Hence, the β-casein isolated from Haflinger mare's milk corresponded to a variant of 226 amino acid residues. The latter was composed by highly multi-phosphorylated isoforms with three to seven phosphate groups, and pIs, determined by 2-DE, ranging from 4.74 to 5.30. Moreover, the equine β-casein was able to deamidate spontaneously, at the level of Asn in the potential deamidation motif 135Asn-Gly136. Approximately 80% of the protein was deamidated after 96 h of incubation under physiological conditions.