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One primiparous and 3 multiparous lactating Holstein cows fitted with ruminal and duodenal cannulas were used in a 4 × 4 Latin square design to determine the efficacy of adding urea to a corn silage-based diet on ruminal fermentation and microbial protein synthesis. Dietary treatments were 0, 0.3, 0.6, and 0.9% urea in diet dry matter (DM); urea was manually top dressed and incorporated into the ration. The basal diet contained (DM basis) 52% forage (with 61% of forage provided as corn silage) and 48% concentrate ingredients. The basal diet was formulated to meet National Research Council (NRC, 2001) requirements for energy and all nutrients except rumen-degradable protein (RDP) and metabolizable protein. Experimental periods lasted 14 d with the first 9 d for adaptation. The basal diet, without urea addition, contained 9.2% RDP in DM and had a predicted RDP balance of −167 g/d (NRC, 2001). There were no effects of dietary treatment on ruminal true digestibility of organic matter or ruminal apparent digestibility of neutral detergent fiber and acid detergent fiber. Total ruminal volatile fatty acid concentrations increased linearly with increasing urea level. Feeding increasing amounts of urea quadratically increased rumen ammonia N concentrations (9.0, 11.9, 12.8, and 17.4 mg/dL at 0, 0.3, 0.6, and 0.9% urea supplementation, respectively), passage of microbial N, and microbial N in duodenal digesta as a percentage of nonammonia N. The results of this study indicate that there were some positive effects of adding urea to the described lactating dairy cow diet, and that microbial protein synthesis was maximized at an average ruminal ammonia N concentration of 12.8 mg/dL when urea was added at 0.6% in diet DM.