The rabbit skeletal muscle T-tubule membranes preparation is the richest source of organic Ca2+ blocker receptor associated with the voltage-dependent Ca2+ channel. Solubilization by 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (CHAPS) in the presence of glycerol leads to a 52% recovery of active receptors as determined by (+)[3H]PN 200-110 binding experiments. The dissociation constant of the (+) [3H]PN 200-110 solubilized-receptor complex was 0.4 +/- 0.2 nM by equilibrium binding and 0.13 nM from the rate constants of association (k1 = 0.116 nM-1 min-1) and dissociation (k-1 = 1.5 10(-2) min-1). The (+) [3H]PN 200-110 receptor has been substantially purified by a combination of filtration of Ultrogel A2 column and lectin affinity chromatography in the presence of trace amount of specifically bound (+) [3H]PN 200-110. The purified material contained polypeptides of apparent molecular weights of 142 000, 32 000 and 33 000. These three components copurified with (+)[3H]PN 200-110 binding activity.