Oocytes segregate chromosomes in the absence of centrosomes. In this situation, the chromosomes direct spindle assembly. It is still unclear in this system which factors are required for homologous chromosome bi-orientation and spindle assembly. The Drosophila kinesin-6 protein Subito, although nonessential for mitotic spindle assembly, is required to organize a bipolar meiotic spindle and chromosome bi-orientation in oocytes. Along with the chromosomal passenger complex (CPC), Subito is an important part of the metaphase I central spindle. In this study we have conducted genetic screens to identify genes that interact with subito or the CPC component Incenp. In addition, the meiotic mutant phenotype for some of the genes identified in these screens were characterized. We show, in part through the use of a heat-shock-inducible system, that the Centralspindlin component RacGAP50C and downstream regulators of cytokinesis Rho1, Sticky, and RhoGEF2 are required for homologous chromosome bi-orientation in metaphase I oocytes. This suggests a novel function for proteins normally involved in mitotic cell division in the regulation of microtubulechromosome interactions. We also show that the kinetochore protein, Polo kinase, is required for maintaining chromosome alignment and spindle organization in metaphase I oocytes. In combination our results support a model where the meiotic central spindle and associated proteins are essential for acentrosomal chromosome segregation. KEYWORDS meiosis; synthetic lethal mutation; homolog bi-orientation; spindle; chromosome segregation; Drosophila C HROMOSOMES are segregated during cell division by the spindle, a bipolar array of microtubules. In somatic cells, spindle assembly is guided by the presence of centrosomes at the poles. In this conventional spindle assembly model, the kinetochores attach to microtubules from opposing centrosomes and tension is established. This satisfies the spindle assembly checkpoint, which then allows the cell to proceed to anaphase (Musacchio and Salmon 2007). Cell division is completed by recruiting proteins to a midzone of antiparallel microtubules that forms between the segregated chromosomes, signaling furrow formation (Fededa and Gerlich 2012;D'Avino et al. 2015). However, spindle morphogenesis in oocytes of many animals occurs in the absence of centrosomes. This may contribute to the high rates of segregation errors that are maternal in origin and are a leading cause of miscarriages, birth defects, and infertility (Herbert et al. 2015). How a robust spindle assembles without guidance from the centrosomes is not well understood. While it is clear that the chromosomes can recruit microtubules and drive spindle assembly (Tseng et al. 2010;Dumont and Desai 2012), how a bipolar spindle is organized and chromosomes make the correct attachments to microtubules is not understood.The Drosophila oocyte provides a genetically tractable system for the identification of genes involved in acentrosomal spindle assembly. Substantial evidence in Drosop...