Abstract Speciation of selenium in garlic harvested in naturally seleniferous soil was investigated. The sample was leached with water and the aqueous extract was fractionated by preparative size-exclusion chromatography. Selenium was found in only one (low-molecular mass) fraction. The chromatographic purity of the fraction was verified by reversed-phase chromatography with inductively coupled mass spectrometric detection. Again, one major signal accounting for more than 95% of the total selenium was observed. The heartcut fraction containing this compound produced an intense peak in an electrospray mass spectrum with the selenium pattern centered at m/z 313 ( 80Se). Protonated molecular ions corresponding to the four Se isotopes gave rich fragmentation patterns by collision induced dissociation that allowed the identification of the selenium species to be γ-glutamyl-Se-methylselenocysteine without the need for an authentic standard.