The mechanism of block of single acetylcholine-activated channels by alkylguanidines was studied by means of the gigaohm seal, patch clamp technique on chick myotubes. The single channel current-voltage relationship in symmetrical Cs solutions was linear over membrane potentials from -100 mV to +100 mV with the reversal potential close to 0 mV. Single channel conductance was estimated to be 40 pS with 120 mM cesium in both external and internal solutions and 54 pS with 360 mM cesium in both solutions. Cs binds within the channel with an apparent KD of approximately 100 mM and the channel has a saturating current of 8 -9 pA at +100 mV. External application of 10 mM and 20 mM methylguanidine decreased single channel current amplitude in a manner dependent upon concentration and membrane potential. The block was greater for inward currents than for outward currents. Ethylguanidine was more potent when applied externally (2 mM) than when applied internally (2-20 mM) and reduced single channel current amplitude in a voltage dependent manner. Increasing internal cesium concentration from 60 to 360 mM decreased the block of the single channel by internally applied ethylguanidine. There appear to be at least two channel binding sites for methyl- and ethylguanidine located approximately 25% of the membrane electric field from either membrane surface. The acetylcholine-activated ionic channel is symmetric with respect to cesium permeation but not to block by ethylguanidine. Permeant ions and blocking ions appear to compete for channel occupancy.