Effects of beta-bungarotoxin (beta-BuTX) on cholinergic transmission were examined in various tissues and species. In the guinea-pig atrial preparation, beta-BuTX in concentrations ranging from 0.01 to 10 microgram/ml abolished the negative inotropic and chronotropic responses to electrical stimulation of the vagus nerve. However, such a blocking action of beta-BuTX was not obtained in the mouse, rat or rabbit atrial preparations. beta-BuTX blocked the neuromuscular transmission in the phrenic nerve-diaphragm preparations of guinea pig, mouse and rat, but the onset was earlier in the guinea pig than in the mouse and rat. Since these results suggest that the guinea pig is the most sensitive to beta-BuTX among the mammalian species so far tested, the effect of beta-BuTX on the cholinergic transmission in various tissues of this species was examined. In the guinea-pig stomach preparation, the cholinergic contractile response to electrical stimulation of the vagal preganglionic nerves was abolished by beta-BuTX, whereas the noradrenergic relaxing response to stimulation of the vagal preganglionic nerve was not affected. In the ileal preparation, the cholinergic contractile response to stimulation of the intramural nerve was little attenuated. These results show that beta-BuTX has not only neuromuscular but also parasympathetic blocking actions and that there are species and regional differences in these blocking actions.