BACKGROUND: During cytokinesis in animal cells, an equatorial actomyosin-based contractile ring divides the cell into two daughter cells. The position of the contractile ring is specified by a signal that emanates from the mitotic spindle. This signal has not been identified and it is not understood how the components of the contractile ring assemble. It is also unclear how the ring constricts or how new plasma membrane inserts specifically behind the leading edge of the constricting furrow. The Rho family of small GTPases regulate polarized changes in cell growth and cell shape by affecting the formation of actin structures beneath the plasma membrane, but their role in cytokinesis is unclear. RESULTS: We have studied the function of two Rho family members during the early cell divisions of Xenopus embryos by injecting modified forms of Rho and Cdc42. Both inhibition and constitutive activation of either GTPase blocked cytokinesis. Furrow specification occurred normally, but ingression of the furrow was inhibited. Newly inserted cleavage membranes appeared aberrantly on the outer surface of the embryo. Microinjected Rho localized to the cortex and regulated the levels of cortical F-actin. CONCLUSIONS: These results show that Rho regulates the assembly of actin filaments in the cortex during cytokinesis, that local activation of Rho is important for proper constriction of the contractile furrow, and that Cdc42 plays a role in furrow ingression. Moreover, our observations reveal that furrow ingression and membrane insertion are not strictly linked. Neither Rho nor Cdc42 appear to be required for establishment of the cell-division plane.