The Bacillus subtilis lutABC operon encodes three iron-sulfur-containing proteins required for Llactate utilization and involved in biofilm formation. The transcriptional regulator LutR of the GntR family negatively controls lutABC expression. The lutP gene, which is situated immediately upstream of lutR, encodes an L-lactate permease. Here, we show that lutP expression can be strongly induced by L-lactate and is subject to partial catabolite repression by glucose. Disruption of the lutR gene led to a strong derepression of lutP and no further induction by L-lactate, suggesting that the LutR repressor can also negatively control lutP expression. Electrophoretic mobility shift assay revealed a LutR-binding site located downstream of the promoter of lutA or lutP and containing a consensus inverted repeat sequence 59-TCATC-N 1 -GATGA-39. Reporter gene analysis showed that deletion of each LutR-binding site caused a strong derepression of lutA or lutP. These results indicated that these two LutR-binding sites can function as operators in vivo. Moreover, deletion analysis identified a DNA segment upstream of the lutP promoter to be important for lutP expression. In contrast to the truncated LutR of laboratory strains 168 and PY79, the full-length LutR of the undomesticated strain RO-NN-1, and probably many other B. subtilis strains, can directly and negatively regulate lutP transcription. The absence or presence of the N-terminal 21 aa of the full-length LutR, which encompass a small part of the predicted winged helix-turn-helix DNA-binding motif, may probably alter the DNA-binding specificity or affinity of LutR.