Aims: The milk acidification rate of Streptococcus thermophilus strains can be affected by several factors, one of which is the hydrolysis of urea by the urease complex. To evaluate the technological suitability of S. thermophilus strains deprived of urease activity in milk fermentation, the genetic cluster related to urease enzymatic activity has been characterized in the type strain DSM 20167(T). Methods and Results: Amplification of the urease genes of S. thermophilus DSM 20167(T) was developed on the basis of the urease gene cluster of the phylogenetically related S. salivarius. Nucleotide sequencing revealed the presence of eight open reading frames, which were most homologous to ureABC (structural genes) and ureI, ureEFGD (accessory genes) of S. salivarius and other ureolytic bacteria. Reverse transcriptase PCR experiments were in agreement with an operon organization for the eight genes (ureIABCEFGD). A food grade mutant A16 (DeltaureC3) with a 693 bp in-frame deletion in ureC gene exhibited a urease negative (Ure(-)) phenotype. Unlike the wild-type strain, the acidification rate of the mutant in reconstituted skimmed milk was not affected by the presence of urea or nickel ions. A small-scale yoghurt fermentation trials were carried out using the wild-type or the Ure(-) mutant A16 (DeltaureC3) in co-culture with Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 in presence of urea. The result obtained underlines that when the Ure(-) mutant was used as a co-starter the acidification rate was higher than that obtained using the wild-type strain. Conclusions: The study provides the first genetic characterization and the technological implication of S. thermophilus DSM 20617(T) urease activity. Significance and Impact of the Study: The detrimental effect of ureolytic activity on the rate of milk acidification was evaluated and superseded using a food-grade Ure(-) recombinant strain. Small-scale yoghurt production trials highlighted the positive role of a Ure(-)S. thermophilus mutant as a co-starter in milk fermentations. Moreover, the vector pMI108 developed for the construction of the Ure(-) strain, should be considered as a potential tool for the generation of Ure(-) dairy S. thermophilus strains selected for other relevant technological properties but characterized by the undesirable ureolytic phenotype.