Four blockers of voltage-gated potassium channels (Kv channels) were tested on the compound action potentials (CAPs) of rat optic nerves in an attempt to determine the regulation of Kv channel expression during the process of myelination. Before myelination occurred, 4-aminopyridine (4-AP) increased the amplitude, duration, and refractory period of the CAPs. On the basis of their pharmacological sensitivity, 4-AP-sensitive channels were divided in two groups, the one sensitive to kaliotoxin (KTX), dendrotoxin-I (DTX-I), and 4-AP, and the other sensitive only to 4-AP. In addition, tetraethylammonium chloride (TEA) applied alone broadened the CAPs. At the onset of myelination, DTX-I induced a more pronounced effect than KTX; this indicates that a fourth group of channels sensitive to 4-AP and DTX-I but insensitive to KTX had developed. The effects of KTX and DTX-I gradually disappeared during the period of myelination. Electron microscope findings showed that the disappearance of these effects was correlated with the ongoing process of myelination. This was confirmed by the fact that DTX-I and KTX enlarged the CAPs of demyelinated adult optic nerves. These results show that KTX- and DTX-sensitive channels are sequestrated in paranodal regions. During the process of myelination, KTX had less pronounced effects than DTX-I on demyelinated nerves, which suggests that the density of the KTX-sensitive channels decreased during this process. By contrast, 4-AP increased the amplitude, duration, and refractory period of the CAPs at all the ages tested and to a greater extent than KTX and DTX-I. The effects of TEA alone also gradually disappeared during this period. However, effects of TEA on CAPs were observed when this substance was applied after 4-AP to the adult optic nerve; this shows that TEA-sensitive channels are not masked by the myelin sheath. In conclusion, the process of myelination seems to play an important part in the regulation and setting of Kv channels in optic nerve axons.