4-Aminopyridine (4AP) and tetraethylammonium ions (TEA), which block voltage-dependent K+ channels in other nerve membranes, were used to study nerve conduction in fibers of normal rat spinal roots and those demyelinated with diphtheria toxin. The pharmacological actions were compared with those of temperature. Both TEA and 4AP increased the amplitude and duration of the monophasically recorded compound action potentials of non-myelinated fibers in normal rat dorsal roots. Enhancement of the action potential amplitude by 4AP was maximal near 1 mM, and was not readily reversed by washing. At concentrations up to 50 mM the action of TEA was weaker and reversible. In normal dorsal and ventral roots TEA (20 mM) and 4AP (5 mM) had only a mildly depressant action on the compound action potentials of myelinated fibers. Whereas the slight reduction in peak amplitude and increase in width was also found in single fibers treated with TEA, none was discerned in single fibers exposed to 4AP over a wide temperature range. Voltage-dependent K+ channels apparently occur in significant numbers in mammalian non-myelinated fibers, but not at nodes of Ranvier. Spinal roots previously treated with diptheria toxin to cause demyelination were studied by longitudinal current analysis. Fibers affected by diphtheria toxin had a late phase of outward current, either restricted to nodes or, in the case of continuous conduction, distributed along internodes, and this outward current was specifically blocked by 4AP. Both 4AP and TEA increased the temperature at which conduction block occurred in most single demyelinated fibers, so that in some cases fibers blocked at physiological temperatures were enabled to conduct. 4AP was more potent than TEA, but less consistent in its effect. K+ channels are apparently present at widened nodes and in internodal axolemma exposed by demyelination. Their presence enables TEA and 4AP to overcome conduction block in some demyelinated nerve fibers.