Milk urea nitrogen (MUN) and blood urea nitrogen are correlated with nitrogen balance and nitrogen excretion; however, there is also a genetic component to MUN concentrations that could be associated with differences in urea transport. It was hypothesized that a portion of the variation in MUN concentrations among cows is caused by variation in gastrointestinal and kidney urea clearance rates. Eight lactating cows with varying MUN concentrations while fed a common diet were infused with [ 15 N 15 N]urea to determine urea N entry rate (UER), gastrointestinal entry rate, returned to ornithine cycle, urea N used for anabolism, urea N excretion in feces and urine. Urea clearance rates by the kidneys and gastrointestinal tract were calculated from isotopic enrichment of urea excretion in urine and gut entry rate, respectively, and plasma urea N concentrations (PUN). Over the course of the experiment, animals weighed an average of 506 ± 62 kg and produced 26.3 ± 4.39 kg of milk/d, with MUN concentrations ranging from 11.6 to 17.3 mg/dL (average of 14.9 ± 2.1 mg/dL). Plasma urea N was positively correlated with UER, urea N excretion in urine, and urea N used for anabolism. Plasma urea N and MUN were negatively correlated with gut clearance rates and ratio of gastrointestinal entry rate to UER. This relationship supports the hypothesis that differences in gut urea transport activity among animals causes variation in PUN and MUN concentrations, and that cows with high PUN and MUN are less efficient at recycling PUN to the gastrointestinal tract and thus may be more susceptible to ruminal N deficiencies when fed low RDP diets. Such biological variation in urea metabolism necessitates an adequate safety margin when setting regulations for maximal MUN levels as an indicator of herd N efficiency., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)