Serum free thyroxine (fT4), thyroxine (T4), and 3,5,3'-triiodothyronine (T3) concentrations were determined in 62 healthy dogs, 51 dogs with hypothyroidism, and 59 euthyroid dogs with concurrent dermatopathy or concurrent illness for which hypothyroidism was a diagnostic consideration. Status of thyroid function was based on history, physical findings, results of thyrotropin response testing, requirement for thyroid hormone replacement therapy, and in 31 dogs, on results of histologic examination of a thyroid gland biopsy specimen. Serum fT4 concentration was determined, using a single-stage radioimmunoassay. Mean (+/- SD) serum fT4 concentration was significantly (P less than 0.05) greater in healthy dogs vs dogs with hypothyroidism (0.51 +/- 0.27 ng/dl vs 0.10 +/- 0.07 ng/dl). Significant difference in mean serum fT4 concentration was not evident between dogs with hypothyroidism and euthyroid dogs with hyperadrenocorticism (0.16 +/- 0.13 ng/dl) or peripheral neuropathy (0.19 +/- 0.10 ng/dl). Mean serum fT4 concentration in all other groups of euthyroid dogs with concurrent illness was similar to values in healthy dogs and was significantly (P less than 0.05) greater, compared with values in dogs with hypothyroidism. Similar results were found for mean serum T4 concentration. Comparison of serum fT4 vs T4 concentration revealed: sensitivity, 0.97 vs 0.98; specificity, 0.78 vs 0.73; predictive value for a positive test result, 0.79 vs 0.80; predictive value for a negative test result, 0.97 vs 0.97; and accuracy, 0.78 vs 0.86, respectively. Ten (17%) and 12 (20%) of 59 serum fT4 and T4 concentrations, respectively, were inappropriately low in euthyroid dogs with concurrent illness.(ABSTRACT TRUNCATED AT 250 WORDS)