In 30 patients anesthetized with halothane and 60 per cent nitrous oxide, and in 12 cats anesthetized with chloralose and urethane,d-tobo-curarine (dTc) was continuously infused to produce constant 90 per cent depression of twitch height prior to injection of neostigmine or pyridostigmine. Mean times from neostigmine, 0.6, 1.2, or 1.8 mg/m2, or pyridostigmine, 3, 6, or 9 mg/m2administration to peak antagonism (onset time) of dTc were 11.1, 8.5, and 7.1 minutes with neo-stigmine and 15.8, 16.9, and 12.2 minutes with pyridostigmine in man. Mean times from administration of the same doses of neostigmine and pyridostigmine to 50 per cent return to the dTc-depressed twitch (duration of action) were 37.8, 41.0, and 57.2 minutes with neostigmine and 51.4, 78.8, and 83.6 minutes with pyridostigmine in man. The onset and duration of action times also were longer with pyridostigmine than with neostigmine in the cat. The doses of pyridostigmine and neostigmine needed for 50 per cent antagonism of the dTc-induced depression of twitch height were 4.0 mg/m2and 0.92 mg/m2for man and 120 μg/kg and 13 μg/kg for the cat, respectively. Thus, the potency ratio of pyridostigmine to neostigmine is 4.35 (4.0/0.92) in man and 9.3 (120/13) in the cat. We conclude that pyridostigmine has a slower onset and longer duration of action than neostigmine.