A plasmid, designated pSUW100, encoding the D(-)lactate dehydrogenase [D(-)-LDH; NAD+ oxidoreductase, EC 1.1.1.28] from Lactobacillus helveticus CNRZ32 was identified from a genomic library by complementation of Escherichia coli FMJ39. The D(-)LDH gene was localized by Tn5 mutagenesis and subcloning to a 1.4-kb region of pSUW100. A 2-kb DraI fragment of pSUW100 encoding D(-)LDH activity was subcloned and its nucleotide sequence determined. Analysis of this sequence identified a putative 1,014-bp D(-)LDH open reading frame that encodes a polypeptide of 337 amino acid residues with a deduced molecular mass of 38 kDa. The distribution of homology to the CNRZ32 D(-)LDH gene in several lactic acid bacteria was determined by Southern hybridization using an internal fragment of the D(-)LDH gene as a probe. Hybridization was detected in leuconostocs and pediococci but not in lactococci or Lactobacillus casei. An integration plasmid was constructed from pSA3 and a 0.60-kb internal fragment of the D(-)LDH gene. This plasmid was used to construct a D(-)LDH-negative derivative of L. helveticus CNRZ 32 by gene disruption; this derivative was determined as producing only L(+)lactic acid. No significant difference in growth or total lactic acid production was observed between CNRZ32 and its D(-)LDH mutant.