Rho family small GTPases have been shown to be involved in various cellular activities, including the organization of actin cytoskeleton in eukaryotic cells. There are six rho genes in the fission yeast Schizosaccharomyces pombe. Cdc42 is known to control the polarity of the cell. Rho1, Rho2 and Rho3 play important roles in controlling cell shape and septation. On the other hand, Rho4 and Rho5 have not yet been characterized. Here we report the function of rho4+ in fission yeast. Gene disruption revealed that rho4+ is not essential for cell growth. However, rho4-null cells were abnormally elongated and had multiple septa of irregular shape at 37 degrees C. In these cells, F-actin patches were randomly localized all over the cell periphery, and cytoplasmic microtubules (MTs) were misoriented. On the other hand, the exogenous expression of a constitutively active Rho4-G23V or Rho4-Q74L in wild-type cells induced depolarization of F-actin patches and cytoplasmic MTs. Rho4 was localized to the cell periphery during interphase and septum during mitosis. Both the binding of GTP and isoprenylation of its C-terminus were necessary for the localization. Furthermore, the localization of Rho4 was likely to be controlled by Rho GAP and Rho GDI. Rho4 may control cell morphogenesis and septation by regulating both the actin cytoskeleton and cytoplasmic MTs.