Drosophila neuroblasts are a model system for studying asymmetric cell division. Neuroblasts bud off a series of smaller progeny, called ganglion mother cells (GMCs). An essential regulator of GMC development is the Prospero homeodomain transcription factor: Prospero is asymmetrically localized to the basal cortex of the mitotic neuroblast and partitioned into the newborn GMC. Prospero is translocated into the GMC nucleus, where it is necessary to establish GMC-specific gene expression. Cortical localization of Prospero protein is observed only during mitosis; cortical localization requires entry into mitosis and cortical delocalization requires exit from mitosis. The tight correlation and functional requirement between mitosis and cortical Prospero localization suggests that mitosis-specific posttranslational modifications may be involved in regulating Prospero subcellular localization. Here we use monoclonals recognizing the N-terminal or C-terminal region of Prospero to explore its posttranslational regulation. One- and two-dimensional Western analysis reveal a complex pattern of Prospero isoforms; phosphatase assays show that there are several phosphoisoforms of Prospero. Developmental 2D Western blots, cell fractionation assays, and analysis of a missense prospero mutation show that cortical Prospero protein is highly phosphorylated compared to nuclear Prospero protein. Our results are consistent with two functions of Prospero phosphorylation: (i) phosphorylation may be required for Prospero cortical localization; or (ii) phosphorylation may be a consequence of Prospero cortical localization, in which case it may facilitate subsequent events, such as Prospero cortical release or nuclear localization.