Adult horses showed a mild diurnal variation in equine plasma thyroxine (T4) concentrations, but not triiodothyronine (T3). Plasma T4 concentrations tended to be higher between 5 PM and 8 PM than at 8 AM. Increases in plasma T4 and T3 were similar in adult healthy horses given 5, 10, or 20 IU of thyroid-stimulating hormone (TSH). The T4 peaked at approximately twice (2.0 +/- 0.4 times) as high as the base line at 6 to 12 hours after the TSH was given. The greatest change from base line T3 occurred at 1 to 3 hours after the TSH was given, but the magnitude of increase was widely variable (4.36 +/- 2.49 times as high as base line). The following method for doing the equine TSH-response test was suggested: (i) prepare plasma or serum sample for determining base line T4 and T3, (ii) inject 5 IU of TSH IM, (iii) prepare plasma or serum samples at 3 and 6 hours after the TSH was injected, and (iv) freeze samples at -20 C until T4 and T3 determination by radioimmunoassay. Treatment of horses with phenylbutazone for 5 days caused a significant decrease in base line T4 and T3 in horses (P less than 0.05). However, phenylbutazone-treated horses responded to the injection of TSH, and the increase in T4 at 6 hours was greater than in the controls (not given phenylbutazone) (P less than 0.02).