Radioimmunoassay kits were validated for triiodothyronine (T3), thyroxine (T4), and hydrocortisone (cortisol) in canine, feline, and equine sera. In each assay, serial dilutions of serum inhibited binding of radioiodinated hormone to the antibody in a manner parallel to inhibition by standard solutions. Parallel inhibition curves indicated that the same substance of standard solutions and serum inhibited binding and that the assays were specific. Further examination of specificity indicated that the antibodies minimally cross-reacted with other compounds structurally similar to T3, T4, and hydrocortisone. A 3rd test for specificity was to show that concentrations of the hormones, as determined by the radioimmunoassays, were consistent with what is known about the hormones' physiologic features. Accuracy was demonstrated by showing that essentially 100% of T3, T4, and hydrocortisone added to samples of serum was measured by the respective assays. Interassay coefficients of variation used to measure precision on 5 quality control specimens ranged from 8.5% to 14.0% for T3, 4.0% to 16.4% for T4, and 10.2% to 16.5% for hydrocortisone. Intra-assay coefficients of variation for all assays ranged from 4.2% to 12.8%. The smallest amounts of T3, T4, and hydrocortisone (sensitivity) that could be precisely quantified were 0.14 ng/ml, 0.17 microgram/dl, and 0.66 microgram/dl, respectively. Reagents and data concerning assay validity should always be described or cited so that the reliability of the radioimmunoassays used can be evaluated.