1. The contributions of specific residues in gamma- and epsilon-subunits to the developmental changes in conductance and open time of Xenopus muscle acetylcholine receptors (AChRs) were investigated. This study was directed primarily at residues in the M2 domains of gamma- and epsilon-subunits; however, the results of additional mutations in the extracellular region flanking M2 and in the amphipathic region between M3 and M4 are also described. 2. The M2 domains of gamma- and epsilon-subunits differ at only three amino acid residues, two of which are adjacent to each other and located near the narrowest part of the pore. These two residues (NI in gamma, SV in epsilon) were found to be major determinants of the difference in conductance and open time of AChRs bearing gamma- or epsilon-subunits. 3. Mutation of N to S in the gamma-subunit converted the long open time of receptors bearing the gamma-subunit (gamma-AChRs) to the brief open time characteristic of receptors bearing an epsilon-subunit (epsilon-AChRs). Conversely, epsilon-AChRs with SV mutated to NI in the epsilon-subunit exhibited a long open time characteristic of gamma-AChRs. 4. Mutation of N to S in the gamma-subunit increased the conductance of gamma-AChRs but did not confer the full conductance of wild-type epsilon-AChRs. Conversely, mutation of SV to NI in the epsilon-subunit reduced the conductance of epsilon-AChRs, but not completely to the level of wild-type gamma-AChRs.