In study 1, four cows had a ruminal canula, a catheter in the right ruminal vein and an ultrasonic flow probe around the right ruminal artery; a catheter was placed in the auricular artery on experimental days. Blood samples were taken every 10 min from -20 to 60 min after ruminal infusion of 5.79 mmol pteroylmonoglutamic acid and cyanocobalamin. There was a net release of these vitamins across the rumen wall following the infusion (P=0.06). In studies 2 and 3, four cows had catheters in the portal, one hepatic and two mesenteric veins and one mesenteric artery. Plasma flow was determined using p-aminohippurate. In study 2, blood samples were taken before and every 30 min for 6 h after feeding 0 or 4 mg of pteroylmonoglutamic acid. Flow of folates through the portal-drained viscera (PDV) and the total splanchnic tissues (TSP) tended to increase with the ingestion of pteroylmonoglutamic acid (P=0.19). In study 3, blood samples were collected every 30 min for the first 3 h to calculate plasma flow and basal net fluxes of folates and vitamin B12. The cows were fed 2.6 g pteroylmonoglutamic acid and 500 mg cyanocobalamin; blood samples were taken every 2 h for 24 h. Vitamin supplements increased the net release of folates and vitamin B12 from PDV (P=0.04) and TSP (P=0.13). The present results demonstrate that, in dairy cows, at doses reported to improve animal performance, passage of pteroylmonoglutamic acid to the portal blood appears during the 6 h following its ingestion, whereas for cyanocobalamin, it is a slow process, not yet completed 24 h after its ingestion.