Prothymosin a is a small, acidic, ubiquitous protein, thought to play a role in cell proliferation, carcinogenesis and apoptosis. We have reported earlier that in the interphase nucleus prothymosin a exhibits a punctuated nuclear distribution associated with transcription sites. Moreover, the protein was found to localize in 1-6 subnuclear domains where PML and CstF64 proteins were also identified. In the present study we followed the subcellular distribution of prothymosin a during mitosis. Our data identify prothymosin a to colocalize with alpha-tubulin in the mitotic spindle throughout mitosis, and to decorate the midbody during cytokinesis. Moreover nocodazole treatment disrupted prothymosin alpha and alpha-tubulin colocalization at the centrosomes of the interphase cell. Prothymosin a was also found to decorate gamma-tubulin identified centrosomes, during mitosis. Taken together our colocalization study suggests involvement of prothymosin a, in the formation, maintenance, or functioning of the mitotic spindle.