The objective of this experiment was to quantify intakes, duodenal flows, and ruminal apparent synthesis (AS) of B-vitamins in lactating dairy cows fed diets varying in forage and nonfiber carbohydrate (NFC) contents. Eight (4 primiparous and 4 multiparous) ruminally and duodenally cannulated Holstein cows were assigned to 4 dietary treatments in a replicated 21-d period, 4 x 4 Latin square design with a 2 x 2 factorial treatment arrangement. Diets, fed as TMR, contained (DM basis) 2 levels of forage (35 and 60%) and 2 levels of NFC (30 and 40%). The forage portion of the diets contained 50% corn silage, 33% alfalfa hay, and 17% grass hay. Soybean hulls and beet pulp (2:1) and corn meal and ground barley (2:1) were included to achieve desired NFC concentrations. No supplemental B-vitamins were fed. B-vitamin AS was calculated as the amount of a specific B-vitamin flowing to the duodenum minus its daily orts-corrected intake. Dry matter and organic matter intakes were higher for cows fed the 35% forage diets and the 40% NFC diets. Increasing dietary forage content decreased ruminal AS of pyridoxine, folic acid, and B12. Increasing dietary NFC content increased ruminal AS of nicotinic acid, nicotinamide, niacin, pyridoxal, B6, and folic acid but decreased AS of B12. Across diets, amounts of B-vitamins synthesized were highest for niacin, followed by riboflavin, B12, thiamin, B6, and folic acid. Biotin AS values were negative for all diets, suggesting either no ruminal synthesis or that destruction by ruminal microflora was greater than synthesis. B-vitamin intake, duodenal flow, and ruminal synthesis are influenced by dietary forage and NFC contents.