An enzyme immunoassay (EIA) was developed for detection of dexamethasone in equine blood. Dexamethasone 21-hemisuccinate-bovine serum albumin was used for immunization of rabbits, and prednisolone 21-hemisuccinate-horseradish peroxidase was used as enzyme conjugate. The assay had sensitivity in the low-picogram range (detection limit, 0.3 pg/well, 50% inhibition of binding at 4.5 +/- 0.7 pg/well). Apart from cortisol, which was recognized by the antiserum at concentration > 8.5 ng/ml, the dexamethasone antiserum failed to interfere with endogenous steroids, but cross-reacted with triamcinolone, flumethasone, and betamethasone. Thus, the antiserum was used to perform simultaneous screening for these synthetic glucocorticoids and to confirm their identity by combining reverse-phase high-performance liquid chromatography (RP-HPLC) and EIA. The immunoreactivity obtained by direct serum measurements was characterized by means of 2 independent RP-HPLC systems. Serum extracts were submitted to RP-HPLC systems I and II, and the fractions were tested by EIA. Immunoreactive peaks were identified by comparing their retention time with that of the standard glucocorticoids used for calibration. Coinjection of an internal standard (methylprednisolone) in RP-HPLC system II yielded reproducible relative retention times. The effectiveness of the test system was evaluated, using blood from a horse treated with commonly used veterinary preparations of dexamethasone. Administration of the free alcohol of dexamethasone and of dexamethasone 21-trioxaundecanoate, both given IV, was detected, and the identity of each was confirmed for up to 48 hours. Intramuscular administration of dexamethasone 21-isonicotinate was continued for at least 14 days after injection of a therapeutic dose. The technique provided higher sensitivity and practicability than do analytic techniques currently available for glucocorticoid testing in horses and proved reliable in confirming the identity of dexamethasone, triamcinolone, flumethasone, and betamethasone in equine blood samples.