The effects of decreasing ruminal urea infusion in lactating dairy cows fed a basal diet deficient in rumen degradable protein on inter-organ urea-N fluxes, epithelial urea-N extraction, and renal urea-N kinetics were investigated. Eight Danish Holstein cows fitted with a ruminal cannula and permanent indwelling catheters in the major splanchnic blood vessels and the gastrosplenic vein were used. The cows were randomly allocated to a triplicate incomplete 3 x 3 Latin square design with 14-d periods. Treatments were continuous ventral ruminal infusion of water, 4.1 g of feed urea/kg of dry matter intake, and 8.5 g of feed urea/kg of dry matter intake. Dry matter intake and milk yield decreased linearly with decreasing urea infusion. Arterial blood urea-N and ruminal ammonia concentrations decreased linearly with decreasing urea infusion. In absolute amounts, the urea-N recycling did not increase when urea infusion was decreased. Arterial urea-N extraction across the portal-drained viscera and rumen wall increased linearly with decreasing urea infusion (2.46, 3.65, and 4.32 + 0.31% and 7.5, 11.5, and 16.9 + 0.9%, respectively), indicating that cows responded to the changes in N supply. The relative urea-N extraction across the ruminal wall increased compared with the total portal-drained viscera extraction. We observed a postprandial decrease in ruminal extraction of arterial urea-N that might reflect that the activity of the protein, presumably facilitating urea-N transport, is regulated by ruminal ammonia. The urea-N clearance by the kidneys decreased (35, 30, and 25 + 2 L/h) and the urea-N reabsorbed by the kidney increased (42, 51 and 56 + 3%) with decreasing urea infusion, indicating that the kidneys salvaged urea-N with low-N supply. The urea transporter B mRNA abundance in rumen papillae (papillae harvested at sampling days) was not affected by dietary N supply. The study showed, that rumen wall extraction of arterial urea-N is subjected to both long-and short-term regulation. Extraction increases with decreasing N supply long-term; however, a short-term postprandial decrease in extraction was observed. No association between long-term adaptation of urea-N extraction across the rumen wall and urea transporter B mRNA abundance could be demonstrated.