The effects of colchicine and cytochalasin B have been investigated on the extrajunctional acetylcholine (ACh) receptors of rat muscle. 2. Colchicine and cytochalasin B each decreased the amplitude and rise time of the iontophoretic ACh potential. When the dose of ACh was increased in the presence of these agents, ACh potentials were produced with a biphasic waveform consisting of a fast initial phase and a second slow phase. 3. It is proposed that colchicine and cytochalasin B are acting through a mechanism other than the breakdown of microtubules and microfilaments.