The objective of this study was to evaluate the effects of substitution of dried distiller grains with soybean meal on beef cattle diets in ruminal ammonia and volatile fatty acid (VFA) concentrations. Red Angus steers (n = 4; BW = 360 ± 9 kg) with ruminal, duodenal, and ileal cannulas, were used in a 4 × 4 latin square. Diets (TMR) consisted of dry-rolled corn (70%), hay (10%), liquid supplement (5%), and test ingredients (15%). Treatments were (DM basis): 1) 15.0% DDGS (CON); SBM in replacement of 50% of DDGS (SBM50); SBM in replacement of 75% of DDGS (SBM75), and SBM in replacement of 100% of DDGS (SBM100). Each period lasted 18-d, consisting of a 7-d diet adaptation followed by an 11-d collection period. Rumen fluid was collected at -2, 0, 2, 4, 6, 8, 10, and 12h post-feeding on d 16. Ruminal digesta was strained through two layers of cheesecloth and two 10-mL aliquots of ruminal fluid were collected and stored at −20 °C until further analysis of ammonia and VFA concentrations. Data were analyzed as repeated measures using the MIXED procedure of SAS with treatment, sampling time, and their interaction as fixed effects, and animal and period as random effects. No treatment by day interactions (P≥0.84) were observed for ammonia or VFA. Ammonia concentrations were greater (P≤0.01) for SBM75 compared to SBM100 and CON, with intermediate values observed for SBM50. Moreover, ammonia was greater (P≤0.01) at 0h compared to 6, 8, and 12h post-feeding. Acetate was greater (P≤0.01) for SBM75 and SBM100 compared to CON and SBM50. Concentrations of propionate were greatest (P≤0.01) for SBM100 and lowest for CON, with SBM50 and SBM75 being intermediate. Further, propionate was greater (P≤0.01) at 16h compared to 0 and 2h post-feeding, with all other times being intermediate. Isobutyrate was lower (P≤0.01) for CON compared to all other treatments, and greater concentrations (P≤0.01) were observed at -2 compared to 14 and 16h post-feeding. Butyrate was greatest (P≤0.01) in SBM75, followed by CON and SBM50; SBM50 was similar to SBM100. Butyrate concentrations were similar -2 and 0h post feeding, which were less (P≤0.01) than all other times. Isovalerate was greater (P≤0.01) for CON than SBM50 and SBM75, and similar to SBM100. Valerate was greatest (P≤0.01) for SBM75 and SBM100 followed by SBM 50 and CON which had the lowest valerate value. Overall, our results show that SBM75 resulted in greater concentrations of ammonia, acetate, propionate, butyrate, isobutyrate, and valerate compared to CON.