The region of theprtR gene coding for the active site of PrtR proteinase was detected in natural isolates of lactobacilli, previously determined asLactobacillus rhamnosus. This region was present in allL. rhamnosus strains with proteolytic activity. The PCR primers used were constructed on the basis of the sequence of the catalytic domain of theprtR proteinase gene. These primers generated in colony-PCR procedure specific 611-bp product with DNA from natural isolates ofL. rhamnosus. No PCR amplifications using these primers were obtained for closely related bacteria of genusLactobacillus, regardless of their proteolytic activity. In addition, these primers could be used singly or in multiplex PCR together with theLactobacillus genus-specific primers. Compared with the other proteinases within the genusLactobacillus (PrtP, PrtB and PrtH) which retained the activity in cell-free proteinase extracts, PrtR proteinase showed proteolytic activity only underin vivo conditions (whole cells of the producing strains).