Studies were carried out on the development and physiological role of receptors for alpha-bungarotoxin (alphaBT) on chick embryo sympathetic neurons maintained in dissociated cell culture. Neurons from embryos of 13 days incubation (E13) developed alphaBT receptors in vitro with a time course and to a maximum level per cell similar to that previously observed for such neurons in vivo. In vitro receptor development by E11 and E8 neurons was also present, but (in comparison with E13 neurons) reached somewhat lower maximal levels. Receptor development in vitro was not affected by exclusion of non-neuronal cells from the cultures. In the present and in previous studies, binding of alphaBT to chick sympathetic neurons was blocked by a variety of ligands of nicotinic acetylcholine receptors. However, saturating concentrations of toxin were found here to be ineffective in blocking either (a) release of [3H]norepinephrine from the cultured neurons elicited via nicotinic stimulation of acetylcholine receptors or (b) depolarizing responses of the cultured neurons elicited by iontophoretically applied acetylcholine and nicotine. Kinetic studies further revealed that, while the idssociation of alphaBT from the cultured neurons is considerably enhanced in the presence of a cholinergic ligand (100 micrometer nicotine), the rate of this dissociation (t1/2 congruent to 30 min) appears to be too slow to account for the inability of the toxin to block nicotinic responses. Such findings show that chick embryo sympathetic neurons can develop receptors for alphaBT both in vivo and in vitro, but that the toxin does not block activation of their nicotinic acetylcholine receptors. The physiologic nature of specific binding sites for alphaBT on such neurons is thus presently unclear.