Objective-To compare effects of 2 acetylcholinesterase inhibitors on recovery quality of horses anesthetized with isoflurane. Animals-6 horses in phase 1, 7 horses in phase 2A, and 14 horses in phase 2B. Procedures-The study comprised 3 phases (2 randomized, blinded crossover phases in horses undergoing orthopedic procedures and 1 prospective dose-determining phase). In phase 1, horses were anesthetized with isoflurane and received neostigmine or saline (0.9% NaCl) solution prior to anesthetic recovery. Phase 2A was a physostigmine dose-determining phase. In phase 2B, horses were anesthetized with isoflurane and received neostigmine or physostigmine prior to recovery. Objective recovery events were recorded and subjective visual analogue scale scores of recovery quality were assigned from video recordings. Results-Recovery measures in phase 1 were not different between horses receiving neostigmine or saline solution. In phase 2A, 0.04 mg of physostigmine/kg was the highest cumulative dose that did not cause clinically relevant adverse behavioral or gastrointestinal effects. Horses receiving physostigmine had higher mean +/- SD visual analogue scale recovery scores (70.8 +/- 13.3 mm) than did horses receiving neostigmine (62.4 +/- 12.8 mm) in phase 2B, with fewer attempts until sternal and standing recovery. Incidence of colic behavior did not differ among groups. Conclusions and Clinical Relevance-Inhibition with physostigmine improved anesthetic recovery quality in horses anesthetized with isoflurane, compared with recovery quality for horses receiving neostigmine. Inhibition of central muscarinic receptors by inhalation anesthetics may underlie emergence delirium in horses recovering from anesthesia.