The directional movement of many cellular organelles in neurons is dependent on polarized microtubules and direction-specific motor molecules. Microtubules are also thought to mediate the retrograde transport of herpes simplex virus (HSV) in sensory neurons. To define the cellular machinery responsible for retrograde axonal transport of HSV, we have investigated the polarity of microtubules in the peripheral axons of trigeminal ganglion neurons. The long ciliary nerves of rabbits were prepared for a standard “hook assay” of microtubule polarity. Axons in cross-sectioned nerves contained microtubules with almost uniform orientation. The fast-growing, plus ends of these axonal microtubules are located distal to the cell body and the slow-growing, minus ends are directed centrally. To determine the role played by microtubules in the retrograde transport of HSV in these axons, we injected the retrobulbar space of mice with the microtubule-inhibiting drugs colchicine, vinblastine, or nocodazole or with the microfilament inhibitor cytochalasin D and 1 d later inoculated the cornea with HSV. We found that colchicine, vinblastine, or nocodazole reduced by 52–87% the amount of virus recovered from the ganglion 3 d postinoculation, compared to vehicle-treated animals. In contrast, cytochalasin D or beta-lumicolchicine did not significantly reduce the amount of HSV recovered from the ganglion. We conclude that the retrograde axonal transport of HSV from axon endings in the cornea to the trigeminal ganglion cell bodies requires intact microtubules and occurs in a plus-to-minus direction on the microtubules. Our data are consistent with the hypothesis that the retrograde axonal transport of HSV is mediated by a minus end-directed motor molecule, for example, cytoplasmic dynein.