The pharmacokinetics and metabolism of tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) have been examined in the mouse, rat, rabbit, and dog using tritiated drug as a marker. In all four species, tiazofurin, given as a single bolus iv injection, is removed from the circulation in a triphasic manner, with a generally prolonged terminal half-life. In all cases, the mean concentration of unchanged drug prevailing during this terminal phase was well within the cytotoxic range (IC50 vs. P388 cells is 2 microM in vitro). Urinary excretion accounted for between approximately 40 and 90% of the administered dose in all four species, with only minor quantities (less than 3%) of drug-derived radioactivity detected in the feces. The metabolism of tiazofurin was examined in mice and rats: although no evidence was uncovered for hydroxylation of tiazofurin at carbon atom 5 of the thiazole ring, phosphorylation of the drug at its 5'-hydroxyl was demonstrable in nearly every organ of both species, but, liver, striated muscles, and kidney were the only tissues catalyzing the synthesis of thiazole-4-carboxamide adenine dinucleotide to any prominent degree. This synthesis did not appear to be a saturated process, even at doses as high as 8000 mg/m2. Since rodent skeletal muscle accumulated high concentrations of tiazofurin phosphates in vivo, it is suggested that musculature may serve as a reservoir for the drug, and contribute to its rather protracted terminal half-life in plasma.