We have investigated the effect of culture temperature on the growth of Lactococcus lactis subsp. lactis biovar diacetilactis, on its acidifying power, on diacetyl and acetoin production, and also on the activities of the principal enzymes involved in the synthesis of these two compounds. The rates of growth and lactic acid production decreased by a factor of ∼ 2 when the temperature decreased from 30 to 18°C. At 18°C, the maximal concentration of diacetyl (0·3mM) was 1·7 times that at 30°C, while that of acetoin was unchanged (5·2 mM). These results are explained by the behaviour of the principal enzymes involved in pyruvate metabolism. The activities of lactic dehydrogenase and acetolactate synthase varied little for culture temperatures between 18 and 30·C. However, the activity of NADH oxidase for a culture temperature of 18°C was 3·7 times that for 30°C, while that of diacetyl reductase for 30°C was 2·7 times that for 18°C. The net effect of temperature on these two activities was an increase in diacetyl production at lower temperature.