AbstractThe pyruvate metabolism in the lactic acid bacterium Lactococcus lactis was studied in anaerobic cultures under transient conditions. During growth of L. lactis in continuous culture at high dilution rate, homolactic product formation was observed, i.e., lactate was produced as the major end product. At a lower dilution rate, the pyruvate metabolism shifted towards mixed acid‐product formation where formate, acetate, and ethanol were produced in addition to lactate. The regulation of the shift in pyruvate metabolism was investigated by monitoring the dynamic behavior of L. lactis in continuous cultures subjected to step changes in dilution rate. Both shift‐up and shift‐down experiments were carried out, and these experiments showed that the enzyme pyruvate formate‐lyase (PFL) plays a key role in the regulation of the shift. Pyruvate formate‐lyase in vivo activity was regulated both at the level of gene expression and by allosteric modulation of the enzyme. A simple mathematical model was proposed to estimate the relative significance of the regulatory mechanisms involved. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 74: 271–279, 2001.