Cytosol prepared from the hind‐limb muscles of the rat contained factors which increased the acetylcholine (ACh) contractures in intact normally innervated muscles in vitro. Muscles which had been denervated 4‐6 days previously and normally innervated muscles released a factor upon incubation which increased the ACh responses of normal muscles. The normal muscles, however, also released an inhibitor of this effect. The medium obtained after incubation of the muscles contained a variety of hydrolytic enzymes including phospholipase C, acid protease and calcium‐activated neutral protease. The amounts of phospholipase C released by normal and denervated muscles were similar, but approximately four times more of the two proteases was released from the denervated muscles than from normal muscles. The possibility that these hydrolases could be the factors which increased the ACh sensitivity in normal muscles is discussed.