Thyroid function was evaluated in 18 healthy dogs by thyrotropin (TSH) stimulation. Two dose regimens were used in each dog: 0.1 IU/kg body weight of freshly reconstituted lyophilized TSH and 1 IU/dog of previously frozen and stored TSH (up to 200 days), both given intravenously. Blood samples were collected prior to and at four and six hours after TSH administration. Serum was evaluated for total thyroxine concentrations by radioimmunoassay. All dogs were classified as euthyroid on the basis of response to 0.1 IU/kg body weight of freshly reconstituted TSH at four and six hours. The 1 IU dose of TSH, previously frozen for up to 200 days, induced increases in serum total thyroxine concentration over baseline at four and six hours that were not significantly different from those resulting from the use of the higher dose of fresh TSH. In all test groups, there were no statistically significant differences between total thyroxine concentrations at four and six hours post-TSH administration. It was concluded that an adequate TSH response can be achieved with the use of 1 IU of TSH/dog for clinically normal dogs between 29.0 kg and 41.6 kg body weight, even if this TSH has been frozen at -20 degrees C for up to 200 days. Further, blood collection can be performed at any time between four and six hours. Similar studies are needed to evaluate this new protocol in hypothyroid dogs and euthyroid dogs suffering nonthyroidal systemic diseases.