Interspecies differences in dihydropyrimidine dehydrogenase (DPD), the initial and rate-limiting enzyme in pyrimidine degradation, were assessed in cytosol from livers isolated from human, monkey, dog, rat, and mouse. Hepatic DPD activity was measured by an HPLC assay with on-line radioactivity detection, using 14C-5-fluorouracil as a substrate. Activity was highly variable within each species and significant interspecies differences in liver DPD activity were observed. The order of activity was mouse > rat > human > dog > or = cynomolgus monkey > rhesus monkey. These data suggest that careful selection must be made when choosing in vivo models of human DPD for the preclinical development of novel fluoropyrimidine anticancer agents and DPD inhibitors.