Aims: To study the influence of peptides and proteolytic enzymes in the osmotic adaptation of Lactobacillus casei. Methods and Results: Di- and tri-peptides added individually increased the osmotolerance of Lact. casei when grown in a chemically defined medium (CDM) containing NaCl. Growth stimulation and the re-establishment in their presence of plasmid DNA supercoiling (recovery of the linking number) in hyperosmotic medium indicated that they are used as osmocompatible solutes as carnithine a known osmoprotector does. The investigation of the proteolytic system showed that in high osmolarity medium, the cell envelope-associated proteinase (PrtP), and PepX (X-prolyl-dipeptidyl aminopeptidase) increased activity and lost repression by peptides. PepI, an iminopeptidase was also derepressed. PepQ, a prolidase that specifically liberated proline from dipeptides, was almost unaffected. Derepression in the presence of peptides took place at the transcriptional level. However, the twofold activation of PrtP in CDM hyperosmotic medium was essentially through an increase of the apparent V-max of the enzyme. Conclusions: These results strongly suggest a contribution of the proteolytic system peptide supply in the osmotic adaptation. Significance and Impact of the Study: Advances in understanding the role of peptides in the adaptation to high osmolarity particularly involved in dairy processes.