Five ovine alpha s1-casein variants (A-E) were identified in an Italian population sample using gel electrophoresis at alkaline pH, gel isoelectric focusing, two dimensional gel electrophoresis, and immunoblotting with polyclonal antibodies against alpha s1-casein. Each casein sample produced two peaks by fast reversed-phase HPLC. Gel isoelectric focusing and electrospray mass spectrometry were used to demonstrate that the first HPLC peak contained the 191 residue alpha s1-casein molecular species and the second the 199 residue species, in proportions of approximately 20:80. Only in the case of the sample containing alpha s1-casein CE was the method for the separation of the single short and long forms of each variant unsuccessful. Both two dimensional electrophoresis followed by staining with polyclonal antibodies against alpha s1-casein and electrospray mass spectrometry showed a heterogeneity consistent with that expected from a protein chain with three levels of phosphorylation and two different lengths. However, especially for alpha s1-caseins D and E, a further uncharacterized heterogeneity was detected.