The hydrolysis of cefamandole nafate, and the O-formyl ester of cefamandole, to cefamandole was studied in vivo in dogs and normal human subjects. After administration of cefamandole nafate to dogs or humans, the parent compound disappeared rapidly from plasma. Disappearance was slightly faster in dogs (half-life [t1/2], 4--6 min) than in humans (t1/2, 6--9 min). The calculated rate constant for hydrolysis of cefamandole nafate was also higher in dogs than in humans, yielding t1/2 values of 6--7 min and 10--17 min, respectively. The rapid hydrolysis of cefamandole nafate to cefamandole in vivo, combined with the partial hydrolysis of cefamandole nafate in vitro before administration (caused by Na2CO3 in the formulation), resulted in circulating levels of cefamandole nafate lower than those of cefamandole. In humans the disappearance of cefamandole nafate was not significantly altered after administration of large (4.0 g) or multiple (4.0g every 6 hr) doses of cefamandole nafate.