Benzimidazoles are widely used as anthelmintic agents and systemic fungicides. In susceptible organisms, benzimidazoles bind to beta-tubulin and block microtubule polymerization. To further characterize this interaction, site-directed mutagenesis followed by gene replacement was used to change Saccharomyces cerevisiae beta-tubulin residue Phe-167 to Tyr. Consistent with previous studies, this mutation resulted in at least 3-4-fold decreased sensitivity to the benzimidazole derivatives carbendazim and nocodazole. The Tyr-167 mutant was cold sensitive, implying a direct effect on benzimidazole binding rather than a nonspecific increase in microtubule stability. Surprisingly, the mutant had 8-fold increased sensitivity to the derivative benomyl, which is structurally identical to carbendazim except at position 1. This suggests that residue 167 interacts with benzimidazoles in the vicinity of the 1-position.