The substituted cysteine accessibility method (SCAM) was applied to the first membrane-spanning segment (M1) of the mouse-muscle acetylcholine (ACh) receptor beta subunit. One at a time, each residue from betaR219 to betaP247, except betaC233, was mutated to Cys, and the mutant beta subunits were expressed together with wild-type alpha, gamma, and delta in Xenopus oocytes. All 28 mutants yielded functional receptors. The accessibility of the substituted Cys to the methanethiosulfonate (MTS) derivatives, MTS ethylammonium (MTSEA), MTS ethyltrimethylammonium (MTSET), and MTS ethylsulfonate (MTSES), added extracellularly in the absence or the presence of ACh, was inferred from their irreversible effects on ACh-induced current. Three consecutive residues close to the extracellular end of M1, betaF224C, betaY225C, and betaL226C, reacted both in the absence and presence of ACh, and one deeper residue, betaV229C reacted only in the presence of ACh. betaV229C also reacted with 2-aminoethyl-2-aminoethanethiosulfonate (AEAETS) and with 2-hydroxyethyl MTS (MTSEH). The rate constants for the reactions of betaV229C with MTSEA, which permeates the open channel, and with MTSEH, which is uncharged, were independent of membrane potential. The rate constant for the reaction of the doubly positively charged AEAETS, however, was dependent on membrane potential, consistent with the exposure of betaV229C in the open channel. The N-terminal third of betaM1, like that of alphaM1, contributes to the lining of the channel and undergoes structural changes during gating.