Rho GTPases are regulators of signaling pathways that control actin organization and cell polarity processes in all eukaryotic cells. In Schizosaccharomyces pombe, Rho4p is involved in the regulation of septum degradation during cytokinesis. Here we show that Rho4p participates in the secretion of the glucanases Eng1p and Agn1p, which are responsible for the septum degradation. First, eng1+ or agn1+ overexpression suppressed the rho4Δ multiseptation phenotype, and simultaneous overproduction of Rho4p and Eng1p or of Rho4p and Agn1p caused a dramatic lysis. Second, Rho4p was not necessary for Eng1p-mediated glucanase activity as measured in cell extracts; however, rho4Δ cells have a lower level of (1,3)-β-d-glucanase activity in the culture medium. Additionally, Eng1- or Agn1-green fluorescent protein did not properly localize to the septum in rho4Δ cells grown at 37°C. There was a decreased amount of these enzymes in the cell wall and in the culture medium of rho4Δ cells at 37°C. These results provide evidence that Rho4p is involved in the regulation of Eng1p and Agn1p secretion during cytokinesis.A.B.M.-C. was supported by a fellowship from the Spanish Ministerio de Educación. This work was supported by grants BIO-2001-1531 and BIO-2001-2041 from the Comisión Interministerial de Ciencia y Tecnología, Spain, and SA026/02 from Junta de Castilla y León.Peer reviewed