Dried distillers grains with solubles (DDGS) can decrease rumen pH because of their inherent acidity. Two replicated 4 × 4 Latin square experiments were conducted with ruminally fistulated heifers to determine the effects of dietary inclusion and NaOH treatment of DDGS on rumen metabolism. In Exp. 1, dietary treatments were 0%, 20%, 40%, or 60% DDGS on a DM basis. The remainder of the diet was 15% corn silage, 20% vitamin-mineral supplement, and corn (to replace DDGS) on a DM basis. Dry matter intake decreased (linear; P < 0.01) with increasing dietary inclusion of DDGS. Rumen pH was less than 5.3 from 1.5 to 12 h after feeding regardless of dietary DDGS inclusion, and mean rumen pH tended to decrease (linear; P = 0.08) with increasing DDGS. Rumen fluid S(2-) and rumen H(2)S gas concentrations increased (P < 0.01) with increasing DDGS inclusion at all time points postfeeding. At 3 h after feeding, ruminal concentrations of acetate, propionate, and total VFA increased linearly (P < 0.04) with increasing dietary inclusion of DDGS. Acetate to propionate ratio (A:P) ranged from 0.97 to 1.25 and was not affected (P = 0.88) by diet over time. In Exp. 2, dietary treatments were 1) 25% DDGS inclusion, untreated, 2) 60% DDGS inclusion, untreated, 3) 25% DDGS inclusion, treated with 2% NaOH, and 4) 60% DDGS inclusion, treated with 2% NaOH. Dry matter intake decreased (P < 0.01) when 60% DDGS was included in the diet regardless of NaOH treatment. Mean rumen pH was greater (P < 0.01) when NaOH-treated DDGS was fed regardless of dietary inclusion level. There were interactions (P ≤ 0.06) of NaOH treatment by DDGS inclusion by time for mean H(2)S and S(2-) concentrations. These interactions occurred because the magnitude of the response to NaOH treatment was greater for the 60% DDGS diets than for the 25% DDGS diets only from 1.5 to 9 h postfeeding. There were no interactions (P > 0.05) of NaOH treatment by DDGS inclusion on VFA concentrations. Acetate concentration decreased (P = 0.04) in cattle fed 60% DDGS diets at 0 h, but at 3 h postfeeding it tended to increase (P = 0.06). Total lactate was less than 1.30 for all dietary treatments at all time points. Treating DDGS with 2% NaOH may increase rumen pH and decrease H(2)S, thereby alleviating polioencephalomalacia.