The effect of feeding a total mixed ration (TMR) and the effect of protein balance in the rumen (OEB) on milk urea concentration (MUC) was studied. Eighteen Holstein cows were offered three diets in a 3 x 3 Latin square design. Separate feeding (rations I and 3) of maize silage in the morning and prewilted grass silage in the evening was compared with a TMR (ration 2). Rations 1 and 2 provided a normal OEB level (+/-150 g/day), whereas ration 3 a high OEB level (+/-400 g/day). All diets were formulated to supply the same level of true protein digested in the small intestine (DVE) and net energy lactation (VEM). The NWC of the morning milk was higher than that of the evening milk with separate feeding, whereas no difference was observed with the TMR. However, feeding strategy had no significant effect on the mean daily WC, which was 247 and 240 mg/l for ration 1 and 2, respectively. The high daily OEB supply resulted in a NTUC of 331 mg/l, corresponding with an increase in NTUC of 0.33 mg/l per g higher OEB. The ammonia (NH3) concentration in rumen fluid of three fistulated lactating cows was determined for each ration in a Latin-square design (3 x 3). The NH3 concentration reached a maximum about 2 h postfeeding. For the diets with equal OEB, peak NH3 levels were similar. The NH3 concentration took longer to decrease after prewilted grass silage was fed than after feeding maize silage. The highest NH3 concentrations were measured for the diet with the high OEB supply. Average daily rumen pH values were equal for all diets. (C) 2003 Elsevier B.V. All rights reserved.