The present study was undertaken to determine the effects of dietary folic acid (FOL) supplementation on ruminal fermentation, duodenal nutrient flow, serum and milk variables, and on B-vitamin concentration in serum. The study was divided into two experiments: in Exp. 1 the forage to concentrate (F:C) ratio of the diet (DM basis) was 34:66 (high concentrate, HC), while in Exp. 2 the F:C ratio was 66:34 (high forage, HF). In addition, the cows received 0 or 1 g FOL/d. In Exp. 1, two German Holstein cows equipped with cannulas in the dorsal sac of the rumen and in the proximal duodenum were dry and five were lactating (186 +/- 144 days in milk); in Exp. 2 four cows were dry and four were lactating (165 +/- 57 days in milk). In cows fed the HC diet, FOL supplementation decreased the ruminally-fermented organic matter. Thus, less energy was available for ruminal microorganisms, which resulted in a reduced microbial crude protein flow at the duodenum. Feeding the HF diet, FOL supplementation only increased the apparent ruminal digestibility of acid detergent fibre (ADF). With the HF diet, FOL had no influence on the serum levels of glucose, non-esterified fatty acids, beta-hydroxybutyrate, urea, thiamine, riboflavin, pyridoxal-5'-phosphate, pyridoxic acid, pyridoxal, pyridoxine, pyridoxamine, pantothenic acid, nicotinamide or nicotinic acid, whereas supplementing FOL to the HC diet lowered the serum glucose and riboflavin levels. In both experiments, the supplementation of FOL had no effects on milk composition. Folic acid supplementation to both diets increased the concentrations of serum 5-methyl-tetrahydrofolate. However, no beneficial effects to dairy cows were obvious. Therefore, to achieve certain results, studies with a higher number of non-fistulated cows would be necessary.