Aims: To investigate the effect of an absence of aminopeptidase PepS on the growth of Streptococcus thermophilus on different media and at different temperatures. Methods and Results: Using gene interruption, a negative mutant of the Strep. thermophilus CNRZ385 strain was constructed for the aminopeptidase PepS (strain Delta pepS). Checks were first of all made using biochemical assays that the Delta pepS strain lacks the peptide hydrolase activity of aminopeptidase PepS. It was demonstrated that the absence of the aminopeptidase PepS exerted a negative effect on growth whatever the culture medium (M17, chemically defined medium, milk). The role of aminopeptidase PepS in growth was enhanced at a high temperature (45 degrees C vs 37 degrees C). The Delta pepS strain was more resistant to lysozyme than the wild-type strain. Conclusions: We were able to demonstrate that aminopeptidase PepS probably plays a pleiotropic role through its involvement in growth via nitrogen nutrition, as well as via other cellular functions/metabolisms (such as peptidoglycane metabolism). Significance and Impact of the Study: This study constitutes the first report on the role of a member of the M29 MEROPS family of metallopeptidases (http://merops.sanger.ac.uk/).