The development of acetylcholine receptors on Xenopus embryonic muscle cells both in culture and in situ was studied using electrophysiology and freeze-fracture electron microscopy. Acetylcholine sensitivity first appeared at developmental stage 20 and gradually increased up to about stage 31. Freeze-fracture of muscle cells that were nonsensitive to acetylcholine revealed diffusely distributed small P-face intramembraneous particles. When cells acquired sensitivity to acetylcholine, a different group of diffusely distributed large P-face particles began to appear. This group of particles was analyzed by subtracting the size distribution found on nonsensitive cells from that found on sensitive cells. We call this group of particles difference particles. The sizes of difference particles were large (peak diameter 11 nm). The density of small particles (< 9 nm) did not change with development. At later stages (32-36) aggregates of large particles appeared, which probably represent acetylcholine receptor clusters. The size distribution of difference particles was close to that of the aggregated particles, suggesting that at least part of difference particles represent diffusely distributed acetylcholine receptors. Difference particles exist mostly in solitary form (occasionally double), indicating that an acetylcholine receptor can be functional in solitary form. This result also shows that diffuse acetylcholine receptors that have previously been observed with 125I-α-bungarotoxin autoradiography do indeed exist in solitary forms not as microaggregates.