When binding of 125I-alpha bungarotoxin (125I-alpha BTX) to hypothalamic membranes is observed over a wide range of concentrations, 3 binding sites can be identified, with estimated equilibrium dissociation constants (Kds) of 4.1 X 10(-11) M, 6.2 X 10(-10) M, and 9.1 X 10(-7) M for high-, low-, and very-low-affinity interactions, respectively. The densities of the high- and low-affinity sites were similar at 14-21 fmol/mg protein, whereas the very-low-affinity site had approximately 1000 X greater capacity. Association and dissociation kinetics predicted a biphasic binding reaction, with association rate constants of 1.38 X 10(8) M-1 min-1 and 7.53 X 10(7) M-1 min-1 and dissociation rate constants of 5.23 X 10(-3) min-1 and 1.80 X 10(-3) min-1. The presence of Na+ inhibited the binding of 125I-alpha BTX with a half-maximally effective concentration of 22 mM. This decrease in binding was associated with the observation of a single binding site with a Kd of 4.3 X 10(-10) M and a density of 12.1 fmol/mg protein. In competition binding experiments, alpha BTX, curare, nicotine, and quinacrine were the most potent competitors. Acetylcholine competed with 125I-alpha BTX binding at 2 sites with estimated affinities of 3.6 X 10(-8) and 7.4 X 10(-5) M. In the rostral hypothalamus, high-affinity binding of 125I-alpha BTX was localized to the region of the supraoptic nucleus, paraventricular nucleus, suprachiasmatic nucleus, and the nucleus circularis complex. Within magnocellular regions, binding was closely associated with neurophysin-immunoreactive neurons and processes, while in the region of the suprachiasmatic nucleus, the binding was in a perinuclear region surrounding parvocellular neurophysin-immunoreactive neurons.