The cardiopulmonary, behavioral, and pharmacokinetic properties of diazepam were determined in horses. Heart rate, cardiac output, mean pulmonary artery, aortic and right atrial blood pressures, respiratory rate, and arterial pH and blood gas values did not change after IV diazepam (0.05, 0.1, 0.2, 0.4 mg/kg) administration. Increasing doses of diazepam resulted in signs of muscle weakness including fixed stance, muscle fasciculations of the head, neck, and thorax muscles, ataxia, and then recumbency. Dosages of diazepam exceeding 0.2 mg/kg produced mild sedation. Behavioral changes persisted for up to 2 hours with a dosage of 0.4 mg/kg. Plasma concentration-time data in horses given brief (5 min) infusions of diazepam were described by a triexponential equation. Total body clearances of diazepam were between 6.94 and 9.56 ml/min/kg, and the half-life values between 2.52 and 21.6 hours. The volume of distribution at steady state was 1.56 to 2.94 L/kg. N-Desmethyldiazepam was identified by gas chromatography-mass spectrometry following glucuronide hydrolysis of urine. Oxazepam, N-methyloxazepam, and N-desmethyldiazepam, but not diazepam, were detected after glucuronide hydrolysis of urine collected from 1 horse which was given 140 mg of IV diazepam. Only N-desmethyldiazepam was detected in hydrolyzed urine samples collected from other horses given 80 mg of diazepam.