The metabolism of [14C]cefotaxime was studied in vivo in rats, dogs, and humans and in vitro in cells of rats and rabbits. Excretion of radioactivity was similar in all species, and greater than 80% of the dose was recovered in the urine. Approximately one-third of the dose was eliminated unchanged, and the major metabolite was desacetylcefotaxime. Under normal circumstances these two products, both with antibacterial activity, were the only materials detected in the plasma. Two further metabolites, designated M2 and M3, (formerly known as UP1 and UP2, respectively, were observed in canine and human urine. Although M2 and M3 were not present in the plasma of normal animals, they were found in the plasma and bile of nephrectomized rats. Extensive studies have shown that the metabolic pathway follows the route: cefotaxime leads to desacetylcefotaxime leads to desacetylcefotaxime lactone leads to M metabolites. The rate-limiting step is the formation of desacetylcefotaxime lactone. All of these reactions take place in the liver. It is concluded that species differences in the metabolism of cefotaxime are more likely to be quantitative than qualitative and that both rat and dog are suitable species for toxicity studies.