Acetylcholine (ACh) contracture of chronically denervated rat diaphragms were studied under different conditions which affect electromechanical coupling. 1. Acetylcholine induced a transient contracture while the membrane potential and the twitch amplitude remained depressed as long as the drug was present. 2. In Tyrode solution with elevated K concentrations, both ACh contractures and twich contractions were diminished or abolished, whereas caffeine was still able to evoke a contracture. 3. In a Ca-free solution, ACh as well as caffeine became ineffective in producing contractures. 4. Manganese depressed both the ACh and caffeine induced contracture, being more effective in the former case. 5. In hypertonic solution and in preparations, in which the T-tubules were disconnected from the plasma membrane by glycerol treatment, acetylcholine failed to initiate contracture, while caffeine could still produce contractures.