The findings of this study show that Class III beta-tubulin is a component of the mitotic spindle in multiple cell types. Class III beta-tubulin has been widely used as a neuron-specific marker, but it has been detected also in association with breast and pancreatic cancers. In this study, we describe a novel finding of Class III beta-tubulin in a subpopulation of cells in malignant peripheral nerve sheath tumor. The findings of this study also show that Class III beta-tubulin is expressed by normal mesenchymal and epithelial cells (fibroblasts and keratinocytes), two transitional cell carcinoma cell lines, and neurofibroma Schwann cells, as shown by immunolabeling and Western transfer analysis using two different Tuj-1 antibodies that are specific for Class III beta-tubulin. The corresponding mRNA was detected using RT-PCR and whole human genome microarrays. Both antibodies localized Class III beta-tubulin to the mitotic spindle and showed a colocalization with alpha-tubulin. The immunoreaction became visible in early prophase, and the most intense immunoreaction was detected during metaphase and anaphase when microtubules were connected to the kinetochores on chromosomes. Class III beta-tubulin-specific immunoreaction lasted to the point when the midbody of cytokinesis became detectable.