The pharmacokinetics and fate of [3H]trospectomycin sulfate, a novel aminocyclitol antibiotic, were examined in male and female dogs and rabbits. Total radioactivity levels in plasma were associated with unchanged trospectomycin in both dog and rabbit. No unchanged drug was found in rabbit urine. Two radioactive components were found in dog urine; one was indistinguishable from trospectomycin and the other was probably a degradation product. The disappearance of drug from plasma followed a biphasic pattern and was well described by a bi-exponential function with half-lives of 0.4-0.8 and 30-70 hr in the dog and 0.4 and 90-120 hr in the rabbit. There was a large distribution volume (Vss), which indicated some retention of drug by tissues. The clearance (CL) for both animals was within the normal range for glomerular filtration rate. CL and Vss were not different between the sexes in the dog or rabbit. Excretion in both animals was initially rapid (greater than 40% by 4 hr) and mainly by the urinary route (fecal excretion less than 10%). Urinary excretion was not significantly different between the sexes. Over the dose range of 25-100 mg/kg, the plasma pharmacokinetics in the dog were linear. However, the recovery of radioactivity in the urine was significantly reduced at the highest dose. Trospectomycin CL in rat, human (obtained from previous studies), dog, and rabbit was described by the allometric equation, CL (ml/hr) = 132 x M0.91 where M is the body mass in kg.