The gene polo encodes a highly conserved serine/threonine protein kinase that has been implicated in several functions during cell division. Polo-like kinases are important positive regulators of cell cycle progression and have also been implicated in the exit from mitosis through the activation of the anaphase-promoting complex. Several data indicate that Plks are required for centrosome function, bipolar spindle organisation and cytokinesis. The intracellular localisation of Plks reflects their multiple roles in cell division, however, in vivo studies that describe the distribution of this protein during different stages of mitosis have never been performed. In the present work, we report the in vivo distribution of a GFP-POLO fusion protein expressed in stable transformants and analysed during the early embryonic development of Drosophila melanogaster. The GFP-POLO protein can be detected in unfertilised oocytes associated with the centromeric region of chromosomes of the polar body and followed until the formation of mitotic domains in later development. Detailed analysis of the dynamic localisation of GFP-POLO during syncytial mitotic cycles shows the timing of localisation to the centrosomes, centromeres and midbody. The results also indicate that GFP-POLO is present in astral microtubules early in mitosis, accumulates around the nuclear envelope until nuclear envelop breakdown and at metaphase associates to spindle microtubules. These in vivo studies show a highly dynamic association of POLO with multiple compartments of the mitotic apparatus. Furthermore, the wide distribution of the GFP-POLO protein to all compartments of the mitotic apparatus provides a valuable tool for future studies on cell cycle during development.