1. In isolated strips of bovine tracheal muscle the carbamate anticholinesterases, neostigmine and eserine caused similar, slowly-developing, sustained spasms which were concentration-related in the range 10(-7)-10(-4) mol/l; these spasms could be abolished either by withdrawing the anticholinesterase or by addition of atropine (5 X 10(-7) mol/l). 2. Depletion of tissue stores of acetylcholine using hemicholinium-3 with or without electrical stimulation rendered the muscle unresponsive to neostigmine (10(-6) mol/l). Responses to acetylcholine itself were not impaired. 3. A low concentration of neostigmine (10(-8) mol/l) did not cause spasm but enhanced the contractile response of bovine trachealis to acetylcholine, carbachol and histamine. This concentration of neostigmine also increased the muscle's contraction upon exposure to a high-potassium solution, even in the presence of atropine (5 X 10(-7) mol/l). 4. It is concluded that neostigmine and eserine cause spasm not only by preventing breakdown of endogenously released acetylcholine but also by stimulating release of acetylcholine from nerve terminals and by a non-specific enhancement of muscle contraction.