AbstractThe appearance of Lac−variants ofLeuconostoc mesenteroidessubsp.mesenteroides19D was studied in different growth conditions, at 30 and 37 °C, in milk and buffered or unbuffered MRS medium. Growth in milk resulted in greater stability of the lactose phenotype. The highest frequency of Lac−variants (40%) appeared in the culture in which mortality was the highest (unbuffered MRS, 37 °C, 96 h). The growth kinetics and fermentation balances of Lac−variants and of the parental strain were identical. No phospho‐β‐galactosidase activity could be detected. The activity of β‐galactosidase was high after growth in lactose‐containing medium and was low in glucose medium. Glucose totally repressed galactokinase activity. Enzymatic analyses of Lac−variants showed that the Lac−character was associated with the total absence of β‐galactosidase activity, in turn correlated with the loss of the 47.3 kb plasmid. An oligonucleotide probe corresponding to a homologous sequence ofLeuconostoc lactisβ‐galactosidase enabled the β‐galactosidase gene t