The properties of interaction of tetrodotoxin with its receptor site on the voltage-sensitive Na+ channel were analysed in two ways: (a) by titrating Na+ channels with a tetrodotoxin derivative, [3H]ethylenediamine-tetrodotoxin; (b) by studying the physiological properties of interaction of the toxin with its receptor from 22Na flux measurements. Using a variety of cell types in culture, three different kinds of situations were observed. 1. Cells like N1E 115 neuroblastoma, CCl 39 fibroblasts, embryonic chick cardiomyocytes and chick skeletal myotubes only have one family of Na+ channels with high-affinity binding sites (in the nanomolar range) for tetrodotoxin. These Na+ channels are the same ones as those that are activated by the alkaloid and polypeptide toxins that accelerate 22Na+ influx. 2. C9 cells have Na+ channels with low-affinity binding sites for tetrodotoxin. These Na+ channels are also the ones that are activated by alkaloid and polypeptide toxins (the median inhibitory concentration for tetrodotoxin inhibition of 22Na+ influx through these Na+ channels in 300 nM). 3. Rat myotubes that have differentiated in culture in the absence of neuronal influence have both high-affinity binding sites (in the nanomolar range) detected with the tritiated tetrodotoxin derivative and low-affinity binding sites (on the micromolar range) detected by 22Na+ flux experiments. Only low-affinity binding sites correspond to Na+ channels that can be activated with alkaloid and polypeptide toxins.