BACKGROUND Venoruton is a standardised mixture of O-(beta-hydroxyethyl) rutosides (HR) used for the relief of oedema and related symptoms in patients with chronic venous insufficiency. OBJECTIVES. The primary objective was to evaluate the pharmacokinetic parameters, in particular the rate and extent of absorption (bioavailability) of two markers of Venoruton: mono-3'-HR and mono-4'-HR derivatives [glucuroconjugated forms (HG)], analysed in their deconjugated form as O-(beta-hydroxyethyl)-quercetin (HQ): mono-3'-HQ and mono-4'-HQ, and to investigate dose proportionality. A secondary objective was to evaluate the general safety of the different dosages. METHODS In this open, single-dose, randomised, four-way, crossover study, 16 healthy volunteers received four different oral doses of Venoruton powder (0.5, 1, 2 or 4 g). Eighteen blood samples were obtained between 10 min pre-dose and 120 h post-dose. RESULTS Peak plasma concentration (C(max)) and area under the plasma concentration-time curve (AUC) of mono-3'-HQ were or tended to be proportional to the dose between 1 g and 4 g. The dose proportionality could be extended to the 0.5-g dose, although C(max) and AUC were not always estimable at that dose level (due to the low number of data points above the limit of quantification). For mono-4'-HQ, the increase of C(max) and AUC was also or tended to be proportional to the dose over the whole tested range (0.5-4 g). Time to peak concentration of both Venoruton derivatives remained unaffected by the administered dose. The elimination half-life of both molecules was very similar with the three highest doses. It was shorter with the 0.5-g dose but was not accurately estimated (or even not estimable in some subjects) due to the low number of points above the limit of quantification. CONCLUSIONS The bioavailability of both Venoruton derivatives (mono-3'-HQ and mono-4'-HQ) tended to be proportional to the dose. The rate of appearance and the elimination half-life of both molecules were not modified with the administered dose. The different doses of the study medication were safe and well tolerated. Mono-3'-HQ and mono-4'-HQ are therefore new bioanalytic and pharmacokinetic markers for Venoruton.