Shortly after the peripheral CS MTs made contact with the equatorial Clorsulon cortex to initiate CR formation, it became concentrated there in a band. Meanwhile, anillin and Factin abruptly accumulated in a narrow band. We found that the initial recruitment of myosin was independent of MTs, but that the restriction step required MT structures. Interestingly, centralspindlin was dispensable in these steps. Instead, the accumulation of myosin required Orbit. The protein was localized in an overlapping region of peripheral MTs, and concentrated in a band on the equatorial cortex at late anaphase. Although centralspindlin was dispensable for the accumulation of Orbit, the Polo and KLP3A-Feo complex, which is essential for Polo recruitment, was required for Orbit accumulation. However, as orbit mutations of consensus amino acid sequences for phosphorylation by Cdk1 or Polo did not influence its accumulation, it appears that the Polo-mediated regulatory system plays an indirect role in Orbit localization. This protein was also necessary for the maintenance of the CR through its close association with myosin and Factin in the CR. Our present data suggest that Orbit plays an essential role as a connector between MTs and the CR during cytokinesis in Drosophila male meiosis. We then examined whether Rho1 and its activator, Pebble, were required for these processes. It is known that Rho1 activation mediated by centralspindlin triggers Dyphylline F-actin polymerization during cytokinesis. Induction of dsRNA for Rho1 resulted in a complete failure of cytokinesis. We found that myosin II accumulation in the CF was entirely disrupted, although its initial recruitment along the cell cortex was observed. This clearly indicated that Rho1 is dispensable for initial myosin recruitment on the cell cortex but is required for its accumulation at the CF during late anaphase. Pebble, which is known as a local activator of Rho1, also accumulated in single rings in the CF region. Expression of the dsRNA successively provided effective depletion of the Pebble protein.