Sensitive and selective high-performance liquid chromatographic methods for the quantitation of the experimental antidepressant fezolamine and its desmethyl metabolite in plasma and urine have been developed. Both assays are linear between 0 and 500 ng/ml in both plasma and urine and have calculated minimum quantifiable levels of less than 10 ng/ml. Statistical evaluation of analytical parameters under single-blind conditions demonstrated an overall precision within +/- 4% of nominal for both compounds in either biological medium. The overall accuracies of the assays were within +/- 5% of nominal values in urine and +/- 10% of nominal values in plasma. Following intravenous administration of fezolamine fumarate to beagle hounds, a biexponential decline in drug plasma levels was observed with the first phase having a half-life of about 11 min and the second phase about 2.6 h. Peak plasma levels of the metabolite were observed at 2 h. Recovery of the parent drug in urine was less than 5% of the administered dose and less than 1% for the desmethyl metabolite.