The ability of various benzimidazoles (BZs) to bind tubulin under different conditions was assessed by determining their IC50 values (the concentration of unlabeled drug required to inhibit 50% of the labeled drug binding), Ka (the apparent equilibrium association constant) and Bmax (the maximum binding at infinite [BZ] = [drug-receptor]). The ability of unlabeled benzimidazoles--fenbendazole, mebendazole (MBZ), oxibendazole (OBZ), albendazole (ABZ), rycobendazole (albendazole sulfoxide, ABZSO), albendazole sulfone, oxfendazole (OFZ), and thiabendazole--to bind tubulin was determined from their ability to inhibit the binding of [3H]MBZ or [3H]OBZ to tubulin in supernatants derived from unembryonated eggs or adult worms of Haemonchus contortus. The binding constants (IC50, Ka, and Bmax) correlated with the known anthelmintic potency (recommended therapeutic doses) of the BZ compounds except for OFZ and ABZSO whose Ka values were lower than could be expected from anthelmintic potency. The binding of [3H]ABZ or [3H]OFZ to tubulin in supernatants derived from BZ-susceptible and BZ-resistant H. contortus was compared. [3H]ABZ demonstrated saturable high-affinity binding but [3H]OFZ bound with low affinity. The high-affinity binding of [3H]ABZ was reduced for the R strain. Tubulin bound BZ drugs at 4 degrees C with lower apparent Ka than at 37 degrees C.