The present experiment was undertaken to verify if the demand of tissues for pteroylglutamic (folic) acid, evaluated by serum clearance and urinary excretion of folates, is different between multiparous dairy cows in late gestation (five gestating dry cows, 52.6 (SD 8.4) d before parturition), and in early lactation (four lactating non-gestating cows, 18.0 (SD 5.9) d after parturition). On day 1 the cows received one intravenous (i.v.) injection of 50 μg pteroylmonoglutamic acid/kg body weight (BW). Blood samples were taken at 0, 5, 15, 30 min, 1, 2, 4, 8 and 24 h after the i.v. injection. On day 3 the cows received one intramuscular (i.m.) injection of 0.3 mg pteroylmonoglutamic acid/kg BW. Blood and urine samples, as well as urine volume, were taken at 0, 1, 2, 4, 8, 12, 24, 36 and 48 h after the i.m. injection. On days 5, 6 and 7 a daily i.m. injection of 0.5 mg pteroylmonoglutamic acid/kg BW was given in an attempt to saturate tissues with folates. Then the cows received one i.v. (day 8) and one i.m. (day 10) injection of pteroylmonoglutamic acid according to the same procedures described previously for days 1 and 3. On day 12 plasma volume was determined. Before tissue saturation, serum clearance of folates during the 24 h following an i.v. injection was similar for gestating and lactating cows but after tissue saturation serum clearance was slower for lactating than gestating cows (stage x saturation, P = 0.04). The percentage of folates excreted in urine was not affected by the physiological stage (P ≥ 0.6) or the level of tissue saturation (P ≥ 0.5). In conclusion, serum clearance and urinary excretion of pteroylmonoglutamic acid seem to support the hypothesis that, in multiparous cows, although there are no deficiency symptoms, tissue demand for folic acid is high, especially during gestation.