The present study examined the endogenous urinary excretion of purine derivatives (PD; allantoin, uric acid and xanthine plus hypoxanthine) in fed animals. Four Rasa Aragonesa ewes fitted with simple cannulas in the rumen and proximal duodenum were used. Animals were given a lucerne (Medicago sativa) hay diet, as sole feed (A) or supplemented, respectively, with 220 (B), 400 (C), and 550 (D) g rolled barley grain/d following a 4 × 4 random factorial design. Duodenal flow of purine bases (PB) was determined by the dual-phase marker system. 15N was infused continuously into the rumen to label exogenous or microbial PB. Duodenal PB flow and urinary excretion of PD increased with digestible organic matter intake showing a constant recovery of duodenal PB. The isotope dilution of PD in urine samples confirmed the presence of an endogenous fraction, originating from tissues, that increased from 115.2 (SE 5.84) μmol/kg W0.75 for the basal diet to 304.2 (SE 7.6) μmol/kg W0.75 at the highest level of duodenal PB.