The Kaposi's sarcoma herpesvirus (KSHV) open reading frame 74 encodes a G protein-coupled receptor (GPCR) for chemokines. Exogenous expression of this constitutively active GPCR leads to cell transformation and vascular overgrowth characteristic of Kaposi's sarcoma. We show here that expression of KSHV-GPCR in transfected cells results in constitutive transactivation of nuclear factor κB (NF-κB) and secretion of interleukin-8, and this response involves activation of Gα and RhoA. The induced expression of a NF-κB luciferase reporter was partially reduced by pertussis toxin and the Gβγ scavenger transducin, and enhanced by co-expression of Gα and to a lesser extent, Gα. These results indicate coupling of KSHV-GPCR to multiple G proteins for NF-κB activation. Expression of KSHV-GPCR led to stress fiber formation in NIH 3T3 cells. To examine the involvement of the Gα-RhoA pathway in KSHV-GPCR-mediated NF-κB activation, HeLa cells were transfected with KSHV-GPCR alone and in combination with the regulator of G protein signaling (RGS) from p115RhoGEF or a dominant negative RhoA(T19N). Both constructs, as well as the C3 exoenzyme from Clostritium botulinum, partially reduced NF-κB activation by KSHV-GPCR, and by a constitutively active Gα(Q226L). KSHV-GPCR-induced NF-κB activation is accompanied by increased secretion of IL-8, a function mimicked by the activated Gα but not by an activated Gα (Q209L). These results suggest coupling of KSHV-GPCR to the Gα-RhoA pathway in addition to other G proteins.