Urease biogenesis was monitored in the lactic acid bacterium Streptococcus thermophilus during the growth cycle using in-gel detection and a phenol-hypochloride assay. Zymogram analysis, performed in a non-denaturing polyacrylamide gel, enabled visualization of a complex profile of bands whose number and intensity were dependent on the growth phase and culture conditions. The monitoring of urease biogenesis in batch fermentations revealed the onset of enzyme synthesis starting from the mid-exponential growth phase, with a maximum reached during the late exponential phase. Urease activity strongly increased at acidic pH but to a lesser extent when urea and nickel ions were added to the culture medium. When S. thermophilus cells were cultured with pH maintained at a neutral value, urease activity was detectable only in gel with extremely low signals. Evaluation of beta-glucuronidase activity in strain DSM 20617(T) harboring a transcriptional fusion between a DNA fragment containing the putative urease promoter and the gusA reporter evidenced significant expression at neutral pH that strongly increased in an acidic environment. Further experiments carried out on p(ureI)-gusA recombinant strain revealed that expression of ure genes was not affected by carbohydrates, nickel or urea availability. The presence of consistent expression of ure genes at neutral pH and the absence of induction of expression by carbohydrate availability demonstrated that the transcription of ure genes in S. thermophilus is regulated differently compared with that of the closely related S. salivarius. These differences are discussed taking into consideration the different habitats colonized by the two bacterial species.