The urease genes of Streptococcus salivarius 57.1 are tightly repressed in cells growing at neutral pH. When cells are cultivated at acidic pH values, the urease genes become derepressed and transcription is enhanced when cells are growing under carbohydrate-excess conditions. Previously, the authors proposed that the bacterial sugar:phosphotransferase system (PTS) modulated the DNA-binding activity by phosphorylation of the urease repressor when carbohydrate was limiting. The purpose of this study was to assess whether enzyme I (EI) of the PTS could be involved in modulating urease expression in response to carbohydrate availability. An EI-deficient strain (ptsI18-3) of S. salivarius 57.1 was constructed by insertional inactivation of the ptsI gene. The mutant had no measurable PTS activity and lacked EI, as assessed by Western analysis. The mutant grew as well as the wild-type strain on the non-PTS sugar lactose, and grew better than the parent when another non-PTS sugar, galactose, was the sole carbohydrate. The mutant was able to grow with glucose as the sole carbohydrate, but displayed a 24 h lag time and had a generation time some threefold longer than strain 57.1. The mean OD600 attained after 48 h by ptsI18-3 supplied with fructose was 0.16, with no additional growth observed even after 3 d. Urease expression in the wild-type and mutant strains was assessed in continuous chemostat culture. Repression of urease at neutral pH was seen in both strains under all conditions tested. Growth of wild-type cells on limiting concentrations of lactose resulted in very low levels of urease expression compared with growth on PTS sugars. In contrast, under similar conditions, urease expression in ptsI18-3 was restored to levels seen in the parent growing on PTS sugars. Growth under conditions of lactose excess resulted in further derepression of urease, but ptsI18-3 expressed about threefold higher urease activity than 57.1. The results support a role for EI in urease regulation, but also indicate that additional factors may be important in regulating urease gene expression.