Concentrations of serum thyroxine (T4) and 3,3',5-triiodothyronine (T3) were determined in 7 clinically healthy adult dogs before and after administration of freshly reconstituted thyrotropin (TSH) and TSH that had been previously reconstituted and frozen for 1, 2, and 3 months. The 4 TSH response tests were performed at 30-day intervals by collecting blood samples for serum T4 and T3 determinations before and 4 and 6 hours after IV administration of TSH (0.1 U/kg of body weight). Baseline serum concentrations of T4 and T3 were similar at each of the 4 sample collection times over the 3-month period of the study. Mean serum concentrations of T4 and T3 increased significantly (P less than 0.01) over baseline values after administration of freshly reconstituted TSH or TSH that had been previously frozen for 1, 2, or 3 months. Significant difference was not found in the mean post-TSH serum T4 or T3 concentration after injection of freshly reconstituted TSH or TSH that had been previously frozen for 1, 2, or 3 months. In 2 of the 7 dogs, mild reactions--mild ataxia and weakness--were observed during the last of the series of TSH response tests (ie, after IV administration of TSH that had been previously frozen for 3 months). Results of this study suggest that for use in dogs, reconstituted TSH stored at -20 C maintains adequate biological activity for at least 3 months. The ability to store reconstituted TSH for a longer period than the recommended 48 hours represents an economic advantage, because it allows clinicians to perform more TSH response tests per vial of TSH.