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Extracts of 31 leek cultivars were analyzed using liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to determine the distribution of the two most abundant S-alk(en)yl-L-cysteine sulfoxides (ACSOs) in leek, i.e., isoalliin and methiin. The isoalliin concentration of the white shaft and green leaves of the 31 leek cultivars varied from 15 to 53 mg/g dry weight (dw) and from 9 to 45 mg/g dw, respectively, whilst the methiin concentration varied from 3 to 16 mg/g dw and from 1 to 10 mg/g dw, respectively. Leek cultivar and tissue had an effect on the ACSO amounts. Cultivars Artico and Apollo F1 rated highest for the mean isoalliin and methiin concentration, respectively. In general, the whole leek plant of the winter leek cultivars contained a significantly higher ACSO amount than the summer and autumn cultivars. To determine whether this difference was attributed to the cultivar background or time of harvest, ACSOs were also quantitated in nine leek hybrids at four different stages during the next growth season. The amounts of ACSO changed significantly during the growth season, indicating the importance of harvest at specific time moments. Although, there was still an effect of cultivar on the ACSO amounts.