An experiment was conducted to determine whether the susceptibility to ruminal acidosis, as defined through differences in days in milk (DIM), milk production level, and ration composition, influences cow feeding, ruminating, and lying behavior and whether these behaviors change during an acute bout of ruminal acidosis. Eight ruminally cannulated cows were assigned to 1 of 2 acidosis risk levels: low risk (LR, mid-lactation cows fed a 60:40 forage:concentrate ratio diet) or high risk (HR, early lactation cows fed a 45:55 forage:concentrate diet). As a result, diets were intentionally confounded with DIM and milk production to represent 2 different acidosis risk scenarios. Cows were exposed to an acidosis challenge in each of three 14-d periods. Each period consisted of 3 baseline days, a feed restriction day (restricting total mixed ration to 50% of ad libitum intake), an acidosis challenge day (1 h meal of 4 kg of ground barley/wheat before allocating the total mixed ration), and a recovery phase. Feeding, rumination, and standing/lying behavior were recorded for 2 baseline days, on the challenge day, and 1 and 4 d after the challenge day for each cow. Across the study, there were no differences in measures of standing, lying, or feeding behavior between the 2 groups of cows. The HR cows did, on average, spend less time ruminating (491 vs. 555 min/d) than the LR cows, resulting in a lesser percentage of observed cows ruminating across the day (44.6 vs. 48.1%). The acidosis challenge resulted in changes in behavior in all cows. Compared with the baseline, feeding time increased on the first day after the challenge (395 vs. 310 min/d), whereas lying time decreased (565 vs. 634 min/d). Rumination time decreased the first day following the challenge (436 min/d) relative to the baseline (533 min/d), but increased the following day (572 min/d). Fewer cows were observed to be ruminating at a given time on the first day following the challenge as compared with the baseline period. Despite this, on a herd level, numerous observations of the proportion of cows ruminating at any one time would need to be taken to accurately detect an acute bout of acidosis using changes in rumination behavior. Overall, these results suggest that risk of acidosis may have little overall effect on general behavior, with the exception of rumination. Furthermore, an acute bout of acidosis alters behavioral patterns of lactating dairy cows, particularly rumination behavior, and identification of these changes in behavior through repeated measurements may assist in the detection of an acidosis event within a herd.