The difference in urinary excretion of mammalian and plant lignans in rats was determined after oral administration of equivalent doses (25 mg/kg of body weight) of 7-hydroxymatairesinol (HMR), lariciresinol (LAR), matairesinol (MR), and secoisolariciresinol (SECO). Twenty-four hours-urine samples were collected after a single dose and after administration of one dose/day for 10 days. Eight lignans were analysed in urine extracts using a high-performance liquid chromatography-tandem mass spectrometry method showing good sensitivity and repeatability. After a single dose of HMR, LAR, MR, and SECO, the main metabolites were 7-hydroxyenterolactone (HENL), cyclolariciresinol (CLAR), enterolactone (ENL), and enterodiol (END), respectively, but after 10-day exposure ENL was the main metabolite of all the tested lignans, showing a considerably higher excretion than after a single dose. Metabolic transformations of plant lignans into each other could also be observed.