Proteolytic and acidifying properties of Streptococcus thermophilus strains isolated from yoghurt or cheeses were evaluated. Among 30 strains tested, 12 exhibited cell envelope-associated proteinase activity (PrtS+), three displayed a slight PrtS activity (PrtS+/−) and 15 were PrtS−, despite the presence of the corresponding gene (prtS) in eight of them. Sequencing of the prtS gene in four PrtS− and one PrtS+ strains revealed that the absence of PrtS activity in the PrtS− strain probably results from an alteration of the prtS regulation. The strains displaying the highest acidifying capacities were all PrtS+. All but one PrtS+ strains were phylogenetically close, as shown by the sequencing of their rDNA internal transcribed spacer (ITS) 16S-23S. More specifically, the high proteolytic and acidifying capacities are associated with the presence of a type II-ITS.