The hypothesis that acetylcholine (ACh) mediates synaptic transmission was tested with the frog''s spinal cord by investigating the effects produced by abolishing cholinesterase activity by prostigmine (3 or 6 X 10-5 M) and then soaking the cord in strong acetylcholine solns. (up to 6 X 10-3 M or even 1.2 X 10 2 M) in addition. Prostigmine (like eserihe) has no detectable effect on the synaptic potential set up in the anesthetized spinal cord by dorsal root volleys even when they are rapidly repetitive. The strong acetylcholine solns. have about the same depressant action on the synaptic potential as is exerted on the endplate potential by 1/500 times that conc of acetylcholine. In the discussion it is argued that since the cholinesterase will have been virtually inactivated these observations refute the hypothesis that acetylcholine mediates synaptic transmission in the frog''s spinal cord.