The objectives of this study were to determine if phenylbutazone decreased serum thyroxine (TT4) and free thyroxine (FT4) concentrations using radioimmunoassay and equilibrium dialysis techniques in horses, and, if so, an additional objective was to determine the duration of this decreased concentration once phenylbutazone administration was discontinued. Serum TT4 and FT4 concentrations were determined before and after administration of 4.4 mg/kg of phenylbutazone i.v. bid for 5 days. Treatment with phenylbutazone caused a significant decrease in TT4 and FT4 concentrations (P < .05). Serum TT4 concentration significantly decreased after day 4 of treatment and remained significantly below baseline value for 10 days after discontinuing phenylbutazone administration; it returned to a value not different from the baseline value by the 11th day. Serum FT4 concentration significantly decreased after day 4 of treatment and remained significantly below the baseline value for only 1 day after phenylbutazone administration was discontinued; it returned to a value not different from the baseline value by the 3rd day after discontinuation of phenylbutazone. These results indicate that serum TT4 and FT4 should not be used to evaluate thyroid function in horses receiving phenylbutazone. In addition, results should be interpreted cautiously when phenylbutazone has been administered within 2 days (for FT4) or within 10 days (for TT4) of sample collection.