Rho proteins are a branch of GTPases that belongs to the Ras superfamily which are critical elements of signal transduction pathways leading to a variety of cellular responses. This family of small GTPases has been involved in diverse biological functions such as cytoskeleton organization, cell growth and transformation, cell motility, migration, metastasis, and responses to stress. We report that several human Rho proteins including Rho A, Rho C and Rac 1, are capable of inducing apoptosis in different cell systems like murine NIH3T3 fibroblasts and the human erythroleukemia K562 cell line. Since K562 cells are devoid of p53, apoptosis induced by Rho in this system is independent of p53. Rho-dependent apoptosis is mediated by the generation of ceramides, and it is drastically inhibited by ectopic expression of Bcl2, both under in vitro and in vivo conditions. Furthermore, the human oncogenes vav and ost that have been shown to function as guanine exchange factors for Rho proteins, were also able to induce apoptosis under similar conditions. Finally, we also report that the levels of endogenous Rho proteins are increased when U937 myeloid leukemia cells are exposed to apoptosis-inducing conditions such as TNF alpha treatment. Furthermore, TNF alpha-induced apoptosis in these cells is inhibited by expression of a dominant negative mutant of Rac 1 but it is not affected by a similar mutant of Rho A. These results suggest that Rho proteins play an important role in the physiological regulation of the apoptotic response to stress-inducing agents.