The nisA promoter is positively regulated in Lactococcus lactis ATCC 11454 by autoinduction via a two-component NisRK-mediated system. However, induction of this promoter can also occur when introduced into the plasmid-free L. lactis LM0230 during growth in galactose or lactose, independent of the NisRK system. In this study, we also characterized this galactose-mediated induction by determining the nisA start site during growth in galactose, which was identical to the nisA start site upon nisin induction. The region involved in the galactose-mediated induction of the nisA promoter was investigated by directed deletion analysis of a 200 bp region upstream of the nisA promoter in the transcription fusion pDOC99. The induction of the deletion derivatives by galactose and nisin was compared phenotypically using β-galactosidase measurements, and the regions necessary for the induction were determined by sequence analysis. Analysis of these regions revealed two sets of a TCT direct repeat ([TCT-N8-TCT] present at positions (-107 to -94) and (-39 to -26) relative to the transcription initiation site. Disruption of the upstream repeat abolished galactose induction and significantly reduced the nisin induction capacity, suggesting a potential pivotal role for these repeats in transcription induction of the nisA promoter. It was also observed that the galactose-mediated induction was abolished when a plasmid containing the phosphotransferase system (PTS), phospho-β-galactosidase and tagatose pathway genes was introduced into this strain. As this effectively made the Leloir pathway redundant, it points to some component of this pathway as the specific inducer of the nisA promoter.