It has been demonstrated that ACh receptors in Xenopus nerve-muscle cultures migrate in the membrane to the nerve contact area during junction formation (Anderson et al., 1977) and that "diffusion trapping" is the major mechanism for nerve-induced receptor accumulation (Kidokoro and Brass, 1985; Kuromi et al., 1985; Kidokoro et al., 1986). A crucial remaining question is how the nerve induces the trap for randomly diffusing ACh receptors. In this study we examined the effect of various glycosaminoglycans in the culture medium on the nerve-induced receptor accumulation and found that heparin and heparan sulfate partially inhibited nerve-induced receptor accumulation, but similar molecules, chondroitin sulfate type A and type C, did not. By chemical modification of heparin we also showed that N-sulfate residues and a large-molecular-weight molecule are essential for this inhibitory effect. Heparin did not affect ACh receptor clustering (hot-spot formation) in myocytes cultured without nerve. By changing the time and duration of heparin application, we found that heparin was effective in inhibiting nerve-induced receptor accumulation only when it was present in the culture medium during the period that neurites are actively forming contact with muscle membrane.