Members of the Rho family of small GTPases activate the nuclear factor kappaB (NF-kappaB) (Perona, R., Montaner, S., Saniger, L., Sánchez-Pérez, I., Bravo, R., and Lacal, J. C. (1997), Genes & Dev. 11, 463-475). We have investigated whether different members of the family of exchange factors specific for Rho proteins (Dbl family) could activate the transcription factor NF-kappaB and have explored both their specificity under in vivo conditions and the mechanisms involved. Activated forms of Dbl, Ost, and Vav proteins induce NF-kappaB activation. While the activation induced by the Vav oncogen was efficiently inhibited by a dominant negative mutant of Rac1, the corresponding mutant of Cdc42Hs was able to block selectively NF-kappaB activation mediated by Dbl. Finally, mutants of RhoA and Cdc42Hs, but not that of Rac1, inhibited the activation of NF-kappaB by Ost. Thus, under in vivo conditions, different members of the Dbl family are related to specific Rho GTPases for the regulation of NF-kappaB. Activation of NF-kappaB by Rho or Ras proteins is mutually independent. However, there is a link between the NF-kappaB and the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) cascades since a dominant negative mutant of MEKK1 is able to inhibit NF-kappaB activation induced by Rac1 and Cdc42Hs proteins, but not by RhoA. These results indicate that, in mammalian cells, multiple pathways coexist for the activation of NF-kappaB, some of which are mediated by specific members of the Ras and Rho families of small GTPases.