The effects of a rat brain supernatant extract and a partially purified supernatant preparation from bovine brain were determined on the binding of [125I]alpha-bungarotoxin (alpha-BGT) to muscle membranes, as well as to membranes prepared from brain. In agreement with previous work, the supernatant preparations inhibited alpha-BGT binding to brain membranes in a dose-dependent fashion, (Brain Research, 245 (1982) 57-67); however, no significant effect of either of the preparations was observed on the binding of the toxin to muscle membranes. As well, the supernatant preparations did not affect binding of radiolabelled alpha-BGT to muscle cells in culture in competition binding experiments. The effect of long-term incubation of cells in culture with the supernatant preparations was subsequently determined. These studies showed that the binding of [125I]alpha-BGT increased markedly (300%) in the presence of a crude rat brain supernatant preparation, while incubation of the muscle cells in the presence of the partially purified bovine supernatant extract had no significant effect on radiolabelled toxin binding. In contrast, both the rat and bovine brain supernatant preparations significantly decreased (up to 65%) radiolabelled toxin binding to a cultured neuronal cell population, adrenal medullary chromaffin cells. These results suggest that an endogenous factor(s), present in brain extracts, differentially regulates the neuronal as compared to the neuromuscular nicotinic alpha-bungarotoxin binding sites.