Posttranslational microtubule modifications (PTMs) are numerous; however, the biochemical and cell biological roles of those modifications remain mostly an enigma. The Aspergillus nidulans kinesin-3 UncA uses preferably modified microtubules (MTs) as tracks for vesicle transportation. Here, we show that a positively charged region in the tail of UncA (amino acids 1316 to 1402) is necessary for the recognition of modified MTs. Chimeric proteins composed of the kinesin-1 motor domain and the UncA tail displayed the same specificity as UncA, suggesting that the UncA tail is sufficient to establish specificity. Interaction between the UncA tail and alpha-tubulin was shown using a yeast two-hybrid assay and in A. nidulans by bimolecular fluorescence complementation. This is the first demonstration of how a kinesin-3 motor protein distinguishes among different MT populations in fungal cells, and how specificity determination depends on the tail rather than the motor domain, as has been demonstrated for kinesin 1 in neuronal cells.