A high-performance liquid chromatographic method for the direct analysis of the urinary metabolites of di-n-butyl phthalate (DBP) is described. In both rats and hamsters the major urinary metabolite found after treatment with either DBP or mono-n-butyl phthalate (MBP) was MBP glucuronide and not MBP as previously reported. The levels of unconjugated MBP in the urine of animals treated with DBP or MBP were three- to fourfold higher in the rat than in the hamster. However, intestinal esterase activities were comparable in the two species, whereas the activities of testicular beta-glucuronidase were significantly higher in rats compared to hamsters. It is possible that the differences in the concentration of free MBP, a substance known to produce testicular damage directly in the rat in vitro, may account for the lack of injury seen in hamsters after oral treatment with either DBP or MBP.