Conversion of pyruvate to alpha-acetolactate, which is broken down to diacetyl and acetoin, can be catalysed by two alpha-acetolactate synthases in Lactococcus lactis. The enzyme encoded by the als gene (Als) has previously been shown to have a low affinity for pyruvate, which limits the formation of diacetyl. In this study we have expressed from a plasmid the ilvBN genes, which encode the other alpha-acetolactate synthase (IlvBN). This plasmid-directed enzyme expression provided up to 3.6-fold increased product formation in the L-lactis MG1363 and IL1403 backgrounds. Plasmid-based expression of the ilvBN genes, in an IL1403 derivative from which the leu.ilv.ald and flanking genes had been deleted, yielded up to 0.1 mM diacetyl whereas the host strain produced none. In addition, IlvBN, with a K-m value of 8.3 mM, was shown to have a greater affinity for pyruvate than does Als.