SUMMARY1. Rats were treated twice daily for 7 days with neostigmine and the diaphragm was isolated for study of its acetylcholine content, release upon nerve stimulation and the number of receptors in the end-plate.2. While the content of total acetylcholine was unchanged, the release of acetylcholine on stimulation with trains of 500 pulses at 100 Hz every 20 sec was reduced by about 50 %. Five days after the end of neostigmine treatment the release of acetylcholine recovered to normal.3. The total number of acetylcholine receptors in the end-plate as measured from the binding of N, O-di[3H]acetyl z-bungarotoxin was reduced from 2-1 x 107 to 1-2 x 107 per end-plate. 4. The above-mentioned changes are not due to acute pharmacological effects of neostigmine, nor to an immediate effect of cholinesterase inhibition but presumably due to chronic accumulation of acetylcholine at the neuromuscular junction.