Eight single-flow continuous-culture fermenters were used in a completely randomized block design with a 2 x 4 factorial arrangement of treatments to investigate the effects of the feed-to-buffer ratio (F/B) on ruminal fermentation, the diversity and community structure of bacteria, nutrient digestibility, and N metabolism. Four diets with forage-to-concentrate ratios of 70:30 or 30:70 with alfalfa or grass hay as forage were supplied to fermenters twice per day at 2 different F/B (23.5 and 35 g of DM/L). The dilution rate was kept constant (5.3%) among all fermenters by infusing the same volume of buffer. An increase in the total volatile fatty acid (VFA) concentration and a decrease in the average pH were observed with an increased F/B. In addition, the molar proportions of all individual VFA found in fermenters differed, depending on the F/B. A terminal restriction fragment length polymorphism analysis showed that the community structure and diversity of bacteria were highly influenced by the F/B. Both diversity and the number of peaks in the electropherograms were lower in most fermenters receiving diets at a high F/B, whereas the similarity percentage of the bacterial communities across diets was higher as the F/B increased. Moreover, the high reduction of neutral detergent fiber digestibility (15.3% +/- 3.65) in fermenters with high F/B suggested a pH-related decrease in the cellulolytic bacterial community as the F/B increased. The crude protein degradation found in fermenters receiving diets with a high F/B was lower compared with that from fermenters with a low F/B. The VFA concentration and purine bases flow response patterns to diets were similar to in vivo conditions only in the case of fermenters with a low F/B. The results suggested that the community structure and diversity of bacteria, as well as the in vitro fermentation parameters, may be affected by the F/B that is used, most likely through a pH effect. In addition, several fermentation parameters showed different response patterns to diets according to the F/B used. Therefore, the amount of feed supplied to single-flow continuous-culture fermenters in which pH is not under control should be carefully chosen according to the volume of buffer infused for the purpose of simulating ruminal fermentation.