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Chromatofocusing was applied to the isolation of ovine β-Lactoglubulin (β-Lg) genetic variants. Ovine β-Lg A and B, which are known to differ only by a single amino-acid substitution, were separated. Moreover, one component differing from A variant in isoelectric point and relative molecular mass has been isolated in samples containing ovine β-Lg A. Although elution of β-Lgs occurred far from their “normal” isoelectric points, chromatofocusing is a powerful technique effective for both analytical and preparative scale.