Abstract We have investigated the possible mechanisms underlying a developmental decrease in acetylcholine (ACh) receptor mobility in the membrane of cultured, spherical, mononucleate Xenopus embryonic muscle cells (myoballs) utilizing the method of in situ electrophoresis. We observed that between 1 and 4 days in culture, a substantial redistribution of ACh receptors can be induced by the externally applied electric field which resulted in highly asymmetrical ACh sensitivities at the cathode- and anode-facing poles of the cell. Between 5 and 8 days in culture, the extent of ACh receptor redistribution induced by the field declined to a lower level. Pretreatment with cytoskeletal disrupting agents or with a disulfide bond reducing agent before in situ electrophoresis had no effect on 2-day-old cultures but enhanced receptor mobility in 6-day-old cultures. Pretreatment with Ca 2+-Mg 2+-free saline (CMF), which releases cell coat material in other systems, substantially increased receptor mobility when tested on days 2, 6, and 8. On day 6, pretreatment with CMF containing cytochalasin B (CB) and colchicine produced an even greater increase in receptor mobility as compared to treatment with CB and colchicine alone. Our findings suggest that the developmental decrease in ACh receptor mobility is accounted for by at least two different mechanisms: (1) An early-developing, CMF-sensitive restriction possibly mediated by the cell coat; (2) a later-developing restriction possibly dependent on cytoskeletal elements and disulfide linkages. The recovery of high ACh receptor mobility in the older cultures following some of the pretreatments indicates that factors determining ACh receptor mobility can arise from molecular interactions external to the lipid bilayer.