The pharmacokinetics and utilization (flavocoenzyme synthesis) of orally and intravenously administered riboflavin in healthy humans were assessed. After the determination of circadian rhythms of riboflavin concentrations in blood plasma and urine of four males and five females (control period), each of these subjects received three different oral riboflavin doses (20, 40, and 60 mg) and one intravenous bolus injection of riboflavin (11.6 mg). Vitamins were administered in a randomized, cross-over design with 2 wk between each administration. Blood plasma and urine specimens were collected repeatedly over a period of 48 h after each administration. Concentrations of flavocoenzymes and riboflavin were analyzed in blood plasma; riboflavin was assayed in urine. During the control period, a small circadian variation was observed: plasma concentrations and urinary excretion of riboflavin were low during the afternoon (P < 0.05). Pharmacokinetics were calculated using a two-compartment open model. The maximal amount of riboflavin that can be absorbed from a single dose was 27 mg per adult. Half-life of absorption was 1.1 h. First-order rate constants describing distribution and elimination of riboflavin were significantly higher after intravenous than after oral administration (P < 0.01). Release of flavocoenzymes into plasma was low compared with the increase of riboflavin concentrations. 7 alpha-Hydroxyriboflavin was identified in plasma. Clearance data indicated that urinary excretion of riboflavin contributes to one-half of the overall removal of riboflavin from plasma. No sex differences were observed for any of the pharmacokinetic variables (P > 0.05).