Diacetyl is a by-product of pyruvate metabolism in Lactococcus lactis, where pyruvate is first converted to alpha-acetolactate, which is slowly decarboxylated to diacetyl in the presence of oxygen. L. lactis usually converts alpha-acetolactate to acetoin enzymatically, by alpha-acetolactate decarboxylase encoded by the aldB gene. We took advantage of the fact that this enzyme also has a central role in the regulation of branched-chain amino acids, to select spontaneous aldB mutants in an unbalanced concentration of leucine versus those of valine and isoleucine in the medium. Industrial dairy strains of L. lactis subsp. lactis biovar diacetylactis containing point mutations and deletions of aldB were isolated and characterized. Their growth in milk was not affected, but they rapidly accumulated a large amount of alpha-acetolactate instead of acetoin from citrate in milk. Under aerated condition, strains devoid of AldB produced about 10 times more diacetyl than did the parental strains.