The interactions of d-tubocurarine (d-TC) with the ionic channel of the nicotinic acetylcholine receptor were studied by biochemical methods in Torpedo electric organ membranes and by electrophysiological methods on frog sciatic nerve-sartorius muscle preparation. Torpedo membranes were treated with alpha-bungarotoxin to inhibit the acetylcholine receptor sites, then binding of [3H]perhydrohistrionicotoxin to the ionic channel sites was studied and found to be inhibited by d-TC. At 37 degrees C, the Ki of d-TC was 10 microM, and at 22 degrees C it was 100 microM. The affinity of d-TC for the ionic channel sites relative to that of perhydrohistrionicotoxin was constant at temperatures from 2-20 degrees C, but increased at higher temperatures up to 37 degrees C. The peak endplate current amplitude was depressed with 1 to 2 microM d-TC in a voltage-dependent manner, with considerable departure from linearity at 10 and 30 degrees C. The effect of d-TC on spontaneous miniature endplate currents was similar and slightly more potent. The time constant of endplate current decay was decreased by d-TC (1 and 2 microM) at temperatures of 10, 15 and 30 degrees C. The channel lifetime was reduced by d-TC, but channel conductance was unaffected. It is suggested that d-TC interacts with both the acetylcholine receptor sites as well as its ionic channel sites in closed and open conformations.