alpha-Bungarotoxin (BuTX; 5 micrograms/ml) completely blocked the endplate potential and extrajunctional acetylcholine (ACh) sensitivity of surface fibers in normal and chronically denervated mammalian muscles, respectively, in about 35 min. A 0.72 +/- 0.033 mV amplitude endplate potential returned in normal muscle fibers after 6.5 hr. of washout of alpha-BuTX, and an ACh sensitivity of 41.02 +/- 3.95 mV/nC was recorded in denervated muscle after 6.5 hr of wash (control being 1215 +/- 197 mV/nC). A two-step reaction of BuTX with binding sites which may allosterically interact is postulated. Several pharmacologic differences were noted between the ACh receptors at the normal endplate and those appearing extrajunctionally following denervation. In normal innervated muscles exposed to BuTX in the presence of 20 microM carbamylcholine or decamethonium, washout of both drugs restored twitch to control levels within 2 hr. Endplate potentials large enough to initiate action potentials were also recorded in most surface fibers. In contrast, these agents, in much higher concentrations (50 microM), were almost ineffective in preventing BuTX blockade of ACh sensitivity in denervated muscle. Hexamethonium (10 and 50 mM) depressed neuromuscular transmission and blocked the action of BuTX in normal muscle in a dose-dependent fashion. On the extrajunctional receptors, hexamethonium (50 mM) was ineffective in protecting against BuTX. We may conclude that at the normal endplate region there are two distinct populations of ACh receptors, both of which react with cholinergic ligands and BuTX, but that a small population (representing congruent to 1% of the total) reacts with BuTX reversibly. Our findings further suggest a clear distinction between ACh receptors located at the normal endplate region and those of the extrajunctional region of the chronically denervated mammalian muscle.