Propionibacterium freudenreichii is a commercially important microorganism that is used in the production of cheeses, cobalamin (vitamin B(12)), and propionic acid. Although a host-vector system in propionibacteria has been developed, there is little information available on the genetic background of the bacteria. To obtain genetic information to facilitate genetic engineering in propionibacteria, we cloned promoter regions from P. freudenreichii using Escherichia coli as a host at the first screening and a promoter-probe vector, pCVE1, which consists of the cholesterol oxidase (choA) gene from Streptomyces sp. as a reporter gene. Finally, nine clones with strong promoter activities in P. freudenreichii were screened by monitoring the choA gene expression and determining if the nucleotide sequences of the cloned DNA fragment were aligned. The initiation sites of these transcripts were determined by primer extension analysis. The putative consensus sequences corresponding to a -35 and -10 hexamer were found to be specific for P. freudenreichii, but not E. coli or other bacteria. Moreover, a new consensus heptamerous sequence between the -35 and -10 regions, termed the -16 region, was also found. It is possible that the putative consensus heptamer is functional and essential to promoter activity in P. freudenreichii. These results should provide new opportunities for controlled gene expression in P. freudenreichii.