The Rho GTPase family and their effectors are key regulators involved in many eukaryotic cell functions related to actin organization and polarity establishment. Schizosaccharomyces pombe Rho1p is essential, directly activates the (1,3)-β-d-glucan synthase, and participates in regulation of cell wall growth and morphogenesis. Here we describe the characterization of the fission yeast Rho5p GTPase, highly homologous to Rho1p, sharing 86% identity and 95% similarity. Overexpression of the hyperactive allele rho5-G15V causes a morphological effect similar to that of rho1-G15V, but the penetrance is significantly lower, and overexpression of the dominant-negative allele rho5-T20N causes lysis like that of rho1-T20N. Importantly, overexpression of rho5+ but no other rho genes is able to rescue the lethality of rho1Δ cells. Shutoff experiments indicated that Rho5p can replace Rho1p, but it is not as effective in maintaining cell wall integrity or actin organization. rho5+ expression is hardly detected during log-phase growth but is induced under nutritional starvation conditions. rho5Δ cells are viable and do not display any defects during logarithmic growth. However, when rho1+ expression is repressed during stationary phase, rho5Δ cells display reduced viability. Ascospores lacking Rho5p are less resistant to heat or lytic enzymes than wild-type spores. Moreover, h90 mutant strains carrying the hyperactive rho5-G15V or the dominant-negative rho5-T20N alleles display severe ascospore formation defects. These results suggest that Rho5p functions in a way similar to, but less efficient than, Rho1p, plays a nonessential role during stationary phase, and participates in the spore wall formation.S.A.R. was supported by a fellowship from the Spanish Ministerio de Educación. This work was supported by grants BIO-2001-1531 and BIO-2004-00384 from the Comision Interministerial de Ciencia y Tecnología, Spain.Peer reviewed