Aspalathin, a dihydrochalcone and C-glycoside, is the most abundant flavonoid in rooibos (Aspalathus linearis), which is well known as an herbal tea in many countries. Aspalathin appears to have in vitro antioxidative and antimutagenic effects. To understand the effects of aspalathin in the body, research on the absorption in the intestinal tract, metabolism in the body, and identification of circulating metabolites in vivo is required. We investigated the metabolism of aspalathin to identify the parent compound and related metabolites in urine and plasma after orally administering a rooibos extract (16.3% aspalathin by 96 g rooibos extract, which equates to 1.1 kg dried rooibos material), produced from unfermented rooibos plant material, to pigs over a period of 11 days (oral dosage, 157-167 mg aspalathin per kg body weight daily). On days 7 and 11 of the study and days 1 and 2 after termination, urine was collected in 24-hour fractions, and plasma samples were collected at various time points. To our knowledge, this is the first time aspalathin metabolites have been identified in vivo, by presenting evidence of the absorption of aspalathin. Six substances identified in the urine by liquid chromatography-mass spectrometry were identified; these represent aspalathin and the metabolites methylated aspalathin, glucuronidated aspalathin glucuronidated and methylated aspalathin, a glucuronidated aglycone of aspalathin, as well as a metabolite of eriodictyol. The latter compound was methylated and contained 2 glucuronic acid moieties. This study showed that aspalathin can be absorbed by the intestine as C-glycoside as well as being cleaved in an aglycone and sugar moiety. The major metabolite in the enzymatically treated samples was methylated aspalathin. Between 0.1% and 0.9% of the administered dose of aspalathin could be detected in the urine on days 7 and 11 of the feeding study. No metabolites or aspalathin were found in plasma samples. The identification of the metabolites in vivo enables investigations to determine the biological potential of rooibos extracts.