ABSTRACT Proteinase PrtP (EC:3.4.21.96) is a cell envelope proteinase (CEP) highly expressed in the probiotic strain Lactobacillus paracasei BL312(VSL#3) that accounts for its anti-inflammatory properties. The main aim of this work is to understand differences in CEP expression between this strain and L. paracasei BL23. Hence, differences in the regulation by amino acid sources of four proteinase related genes (prtP, prsA, prtR1 and prtR2) were determined by RT-qPCR in BL312(VSL#3) and BL23 using as a reference BL368, a BL23 derepressed mutant lacking the response regulator (RR) PrcR. BL312(VSL#3) showed greater expression of prtP (2- to 3-fold) than BL23, and prtP was highly repressed by peptone in both strains. Two other putative CEP genes, prtR1 and prtR2, showed a low expression profile. Interestingly, when the prsA-prtP promoter region from both strains, and deleted mutants, were cloned in vector pT1GR, expression of the gfp and mrfp fluorescent reporters was always repressed in BL23 (high or low peptone) and derepressed in BL368, revealing an interesting mechanism of regulation affecting specifically to this promoter. In conclusion, BL312(VSL#3) has higher expression of prtP and other CEP related genes than BL23, that could respond to a natural deregulation in this strain, possibly independent from the RR PrcR.