When ruminants are fed high-concentrate diets, Streptococcus bovis proliferates rapidly and produces lactate, potentially causing rumen acidosis. Understanding the regulatory mechanisms of the metabolism of this species might help in developing dietary strategies to alleviate rumen acidosis. S. bovis strain S1 was newly isolated from the ruminal fluid of Saanen dairy goats and then used to examine the effects of glucose and starch on bacterial metabolism and gene regulation of the organic acid-producing pathway in cultures at a pH of 6.5. Glucose or starch was added to the culture medium at 1 g/liter, 3 g/liter (close to a normal range in the rumen fluid), or 9 g/liter (excessive level). Lactate was the dominant acid produced during the fermentation, and levels increased with the amount of glucose or starch in a dose-dependent manner (P <0.001). The production of formate and acetate in the fermentation media fluctuated slightly with the dose but accounted for small fractions of the total acids. The activities of lactate dehydrogenase (LDH) and a-amylase (a-AMY) increased with the starch dose (P <0.05), but the a-AMY activity did not change with the glucose dose. The relative expression levels of the genes ldh, pfl(encoding pyruvate formate lyase), ccpA (encoding catabolite control protein A), and a-amy were higher at a dose of 9 g/liter than at 1 g/liter (P <0.05). Expression levels of pfland a-amy genes were higher at 3 g/liter than at 1 g/liter (P <0.05). The fructose 1,6-diphosphate (FDP) concentration tended to increase with the glucose and starch concentrations. In addition, the S. bovis S1 isolate fermented glucose much faster than starch. We conclude that the quantities of glucose and soluble starch had a major effect on lactate production due to the transcriptional regulation of metabolic genes.