We used six ruminally cannulated steers in a two-period crossover design to study ruminal fermentative and microbial changes associated with induced subacute acidosis. Steers were adapted to either an 80% alfalfa hay (hay-adapted)- or corn grain (grain-adapted)-based concentrate diet. After feed was withheld for 24 h, steers were overfed with an all-grain diet at 3.5 x NEm daily for 3 d. Ruminal contents and jugular blood samples were collected before withholding feed and at 0 and 12 h daily for 3 d during the overfeeding period. Ruminal samples were analyzed for pH, lactate, VFA concentrations, and counts of total anaerobic, amylolytic, lactic acid-producing and -fermenting bacteria, and ciliated protozoa. Blood samples were analyzed to assess acid-base status. Ruminal pH declined to a range of 5.5 to 5.0 with increased VFA concentrations, but normal lactate concentrations (<5 mM) were indicative of subacute acidosis. Total viable and amylolytic bacterial counts were higher (P < .05) in grain-adapted than hay-adapted steers. Anaerobic lactobacilli counts increased over time (P < .01) in both groups and were generally higher in grain-adapted than hay-adapted steers. Lactate-utilizing bacteria were initially greater in grain-adapted than hay-adapted steers and increased over time in both groups following grain challenge. Total ciliates were initially higher (P < .05) in grain-adapted than hay-adapted steers and decreased after 48 h in both groups. Blood acid-base changes were minimal. Bacterial changes associated with subacute acidosis resemble those reported during adaptation to grain feeding, and the decline in ciliated protozoa may be the only microbial indicator of a potentially acidotic condition in the rumen.