The purpose of this work was to investigate whether the antineoplastic activity of usnic acid affects the formation and/or stabilization of microtubules by visualizing microtubules and determining mitotic indices after treatment of human cancer cells. The visualization of microtubules was carried out in breast cancer cell line MCF7 and lung cancer cell line H1299 using fluorescence microscopy. The cells were seeded in glass chamber slides, cultivated in DMEM for 24 h and treated with 29 mM usnic acid, 1 mM vincristine (which prevents the formation of microtubules) or 1 mM taxol (which stabilizes microtubules) for 4 or 24 h. Cells were fixed with methanol and microtubules observed with the antibody anti-a-tubulin. The mitotic index was reported as the percentage of mitotic cells per total number of cells. Although the concentration of usnic acid was above the IC₅₀, no changes were observed in the morphology of microtubules in MCF7 or H1299 even when cells were exposed to usnic acid for considerably longer periods (24 h) than to vincristine or taxol (4 h). The treatment of MCF7 and H1299 cells with usnic acid yielded mitotic indexes of 6% and 4%, respectively, whereas vincristine presented a mitotic index of about 50% after 8h. Therefore, the usnic acid did not arrest the cell cycle at M phase. These results indicate that the disruption of normal metabolic processes in cells triggered by the action of usnic acid does not primarily involve depolymerization or stabilization of microtubules in breast or lung cancer cells. They also suggest that the antitumour activity of usnic acid is not related to alterations in the formation and/or stabilization of microtubules.