Compositional analysis by liquid chromatography/mass spectrometry of triterpenoid glycosides in different tomato cultivars, ripening stages, and parts of fruits showed that alpha-tomatine was generally most abundant in the flesh of the mature green stage, whereas esculeoside A was predominant in that of the red ripe stage. The sum of these glycoalkaloids was more or less constant, suggesting that alpha-tomatine is converted to esculeoside A during ripening. Besides various substitutions, the C22alphaN --> C22betaN isomerization is an important step in this transformation. By quantum chemical calculations it was shown that hydroxylation at C27 of the triterpenoidal skeleton is the driving force behind the isomerization. For the protonated form of the glycoalkaloid (predominant at the pH of tomato tissue), the C22betaN configuration becomes more favorable than that of C22alphaN, through the extra energy provided by the hydrogen bond between the protonated nitrogen and the lone pair of the oxygen of the C27-OH.