A procedure for the determination of hydrocodone (dihydrocodeinone) in serum has been developed. Hydrocodone and N-isobutyldihydronorcodeinone, the internal standard, are extracted from serum by chloroform-isopropyl alcohol (9:1, v/v). The extracts are purified by back-extraction into 0.1 N sulfuric acid and a final basic extraction into benzene. The pentafluorophenylhydrazone derivatives are formed and determined using electron capture gas chromatography. As little as 1 ng/ml of hydrocodone in serum can be determined. A closely related compound and potential metabolite, dihydronorcodeinone, does not interfere. Serum hydrocodone levels were determined in dogs after oral and intravenous doses of 0.5 mg/kg, and in humans after a 10-mg oral dose of the bitartrate. A mean peak serum drug concentration of 23.6 ng/ml and a terminal half-life of 3.8 h resulted from the human study. The terminal half-life in serum was 1.8 h after the intravenous dose in dogs.