Sixteen multiparous lactating Holstein cows (four with rumen cannulae) were fed diets varying in the content and form of ruminally degradable carbohydrates and N to examine dietary effects on microbial protein synthesis (MPS) and whole animal N efficiency, and to evaluate the use of a model based on milk urea N (MUN) for predicting urinary N excretion and N utilization efficiency (NUE). A replicated Latin square design (consisting of diet and experimental period) was employed. The four diets consisted of two low protein diets with either 20% ground corn (diet LP) or 13.5% ground corn plus 3% sucrose (diet LP sucrose) and two high protein diets with 13.5% corn and 3% sucrose with either urea (diet HP urea) or soybean meal (diet HP SBM) as supplemental rumen-degradable protein sources. The intakes of dry matter and N were increased by increasing dietary crude protein (CP) level. However, the yields of milk and milk protein were not affected by CP level. Yield of microbial protein was reduced by sucrose and increased by CP level. There were no differences between urea and SBM supplementation on DM intake, milk yield, or MPS. Mean urinary N excretion for all cows (252 g/d) was underestimated by 55 g/d or overestimated by 25 or 33 g/d using alternative equations based on MUN. Subsequently, NUE (mean = 22.4%) was underestimated by 7.5, 3.2, or 2.9%, using a previously published set of equations. Urinary N excretion and NUE could be predicted within 10 and 14% of observed values, respectively, using a set of equations incorporating MUN. Therefore, MUN appears to be a useful tool to help assess N losses from lactating cows.