Myotomal muscle cells, derived from Xenopus embryos and grown in culture without nerve, develop discrete sites of cholinesterase (ChE) activity on their surface. The spatial relationship of these ChE patches to surface patches of acetylcholine receptors (AChRs) has been examined in the present study by a combination of ChE histochemistry and fluorescent staining of the receptors. ChE patches and AChR patches developed as early as the 1st day in culture and exhibited a high incidence of spatial overlap. The frequency of overlap varied with the age of the culture and ranged from 50 to 98% for patches on the lower cell surface (facing the floor of the culture dish) and from 28 to 79% for patches on the upper cell surface. The high incidence of overlap cannot be explained on the basis of a random distribution of patches since both types of patch occupied less than 3% of the cell surface. ChE and AChR patches also developed when cultures were grown in a serum- free medium as well as when cultures were prepared from young embryos in which muscle innervation had not yet begun. At some patches, the surface membrane was invaginated and at these invaginations, there was also a high incidence of overlap between the ChE and AChR stains. It is concluded that the mechanisms involved in the localization of AChRs and ChE on the surface of Xenopus myotomal muscle cells tend to be closely linked and operate even in the absence of innervation, previous contact by nerve, or electrical and contractile activity. Considered together with previous ultrastructural observations, the present findings suggest that these cells develop elaborate postsynaptic-like specializations even in the absence of neural factors.