The effects of grain-induced subacute ruminal acidosis (SARA) in lactating dairy cows on free ruminal lipopolysaccharide (LPS) and indicators of inflammation were determined. Four mid lactation dairy cows were divided into 2 groups of 2 cows and used in a repeated switchover design. During each period, SARA was induced in 2 animals for 5 subsequent days by replacing 25% of their total mixed ration (dry matter basis) with a concentrate made of 50% wheat and 50% barley. The other 2 cows acted as controls and were fed a total mixed ration diet in which 44% of dry matter was concentrate. On average, inducing SARA did not affect milk composition, increased the duration of rumen pH below 5.6 from 187 to 309 min/d, and increased free ruminal LPS concentration from 24,547 endotoxin units (EU)/mL to 128,825 EU/mL. Averaged across treatments, milk fat yield and milk protein yield were 0.66 and 1.00 kg/d, respectively. Rumen pH and milk fat data suggest that control cows also experienced ruminal acidosis, albeit a milder form of this disease than SARA cows. Serum LPS concentration in both control and SARA cows was less than the detection limit of <0.01 EU/mL for the assay. Induction of SARA elevated serum amyloid A concentration from 286.8 to 498.8 mug/mL, but did not affect other markers of inflammation including haptoglobin, fibrinogen, serum copper, or white blood cells. These results suggest that grain-induced SARA in mid lactation dairy cows increases the lysis of gram-negative bacteria and activates an inflammatory response.