The small GTPase Rho1 has been shown to regulate the organization of the actin cytoskeleton and formation of the cell wall in the fission yeast Schizosaccharomyces pombe. Activity of Rho1 must be precisely regulated in vivo, since both increases and decreases in its activity affect cell growth and shape. Thus, it is important to clarify the mechanism by which the activity of Rho1 is regulated in vivo. Seven genes encoding putative GAPs, GTPase-activating proteins, for the function of the Rho-family proteins were isolated from S. pombe. After disruption of these genes, rga1+ was found to play important roles in cell growth and morphogenesis. In rga1 null cells, delocalized F-actin patches and extraordinary thickening of the cell wall and the septum were observed. On the other hand, over-expression of Rga1 produced shrunken or dumpy cells. The phenotype of the rga1 null cells or the Rga1-over-expressing cells was similar to that of cells containing abnormally high or low Rho1 activity, respectively. Moreover, direct association of Rga1 with Rho1 was shown. Rga1 was localized to the cell ends and septum where Rho1 is known to function. In S. pombe, Rga1 is involved in the F-actin patch localization, cell morphogenesis, regulation of septation, and cell wall synthesis, probably functioning as a GAP for the function of Rho1.