Glycolysis constitutes the primary energy-generating pathway of most species of lactic acid bacteria. The metabolism ultimately results in massive lactic acid production, which is responsible for the major preservative effect of these organisms. This study reports the identification, sequencing, and characterisation of the central glycolytic operon, the gap operon, from Lactobacillus plantarum NC8 and L. sakei Lb790. The structure of the operons of the two Lactobacillus strains were similar and organised in the order cggR-gap-pgk-tpi-eno, encoding a putative central glycolytic gene regulator and the four glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, triosephosphate isomerase, and enolase, respectively. This operon structure has not been reported in any other bacterial species so far. Transcriptional analysis revealed three major transcripts, the mono-cistronic gap and eno and the tetra-cistronic gap-pgk-tpi-eno.