Low ruminal pH may occur when feeding high-concentrate diets. However, because the reduction in pH occurs at the same time as the amount of concentrate fed increases, the changes observed in rumen fermentation may be attributed to pH or the type of substrate being fermented. Our objective was to determine the contribution of pH and type of substrate being fermented to the changes observed in rumen fermentation after supplying a high-concentrate diet. Eight dual-flow, continuous culture fermenters (1,400 mL) were used in 4 periods to study the effect of pH and type of diet being fermented on rumen microbial fermentation. Temperature (39 degrees C), solid (5%/h), and liquid (10%/h) dilution rates, and feeding schedule were maintained constant. Treatments were the type of diet (FOR = 60% ryegrass and alfalfa hays and 40% concentrate; CON = 10% straw and 90% concentrate) and pH (4.9, 5.2, 5.5, 5.8, 6.1, 6.4, 6.7, and 7.0). Diets were formulated to have similar CP and ruminally undegradable protein levels. Data were analyzed as a mixed-effects model considering the linear, quadratic, and cubic effects of pH, the effects of diet, and their interactions. Semipartial correlations of each independent variable were calculated to estimate the contribution of each factor to the overall relationship. True digestion of OM and NDF were affected by pH, but not by type of diet. Total VFA were reduced by pH and were greater in CON than in FOR. Acetate and butyrate concentrations were reduced by pH but were not affected by diet. Propionate concentration increased as the pH decreased and was greater in CON than in FOR. Ammonia-N concentration decreased with decreasing pH and was lower in CON than in FOR. Microbial N flow was affected by pH, diet, and their interaction. Dietary N flow increased as pH decreased and was greater in CON than in FOR. The degradation of CP followed the opposite pattern, increasing as pH increased, and was less in CON than in FOR. The efficiency of microbial protein synthesis (g of N/kg of OM truly digested) was slightly reduced by pH and was less in CON than in FOR. These results indicate that the effects of feeding a high-concentrate diet on rumen fermentation are due to a combination of pH and substrate. Furthermore, the digestion of OM in high-concentrate diets is likely limited by the pH-induced effects on the microbial population activity.