The fungicide carbendazim (CBZ) is known to disrupt microtubular structures in the testis and to cause testicular toxicity in rats. To investigate the mechanism underlying the toxicity of CBZ, tubulin and microtubule-associated proteins (MAPs) were isolated from rat testis and brain using two techniques. The effects of CBZ on MT assembly were compared with the known microtubule (MT) disruptors, colchicine and nocodazole. CBZ (100 μM) had no effect on the assembly of MTs from MAP-containing tubulin isolated with one cycle of glycerol-dependent assembly and disassembly while colchicine (40 μM) and nocodazole (12.5 μM) strongly inhibited the assembly reaction. Similarly, formation of MTs from tubulin prepared with two cycles of glycerol-dependent assembly was strongly inhibited by colchicine and nocodazole but only weakly by CBZ. All three compounds inhibited the assembly of MTs from MAP-free tubulin isolated with glutamate. However, the inhibition by CBZ was reversed by the inclusion of high-molecular-weight MAPs and not by unrelated protein (bovine serum albumin, BSA). Addition of nocodazole to assembled MTs caused immediate depolymerization, whereas CBZ did not directly cause depolymerization. However CBZ was an effective inhibitor of the polymerization of depolymerized tubulin. In competitive binding assays, CBZ was found to inhibit the binding of guanosine triphosphate (GTP) to tubulin. The data suggest that CBZ interferes with initial events of MT polymerization, specifically GTP binding, and that MAPs moderate this effect.