Schizosaccharomyces pombe Rho1p is essential, directly activates beta-1,3-glucan synthase, and participates in the regulation of morphogenesis. In S. pombe, Rho1p is activated by at least three guanine nucleotide exchange factors (GEFs): Rgf1p, Rgf2p, and Rgf3p. In this study we show that Rgf2p is a Rho1p GEF required for sporulation. The rgf2+ deletion did not affect forespore membrane formation and the nuclei were encapsulated properly. However, the mutant ascospores appeared dark and immature. The rgf2Delta zygotes were not able to release the ascospores spontaneously, and the germination efficiency was greatly reduced compared to wild-type (wt) spores. This phenotype resembles that of the mutants in bgs2+, which encodes a sporulation-specific glucan synthase subunit. In fact, glucan synthase activity was diminished in sporulating rgf2Delta diploids. Rgf2p also plays a role in beta-glucan biosynthesis during vegetative growth. Overexpression of rgf2+ specifically increased GTP-bound Rho1p, caused changes in cell morphology, and elicited an increase in beta-1,3-glucan synthase activity. Moreover, the simultaneous disruption of rgf1+ and rgf2+ was lethal and both Rgf1p and Rgf2p were able to partially substitute for each other. Our results suggest that Rgf1p and Rgf2p are alternative GEFs with an essential overlapping function in Rho1p activation during vegetative growth.