Gα 12, a member of α subunit of heterotrimeric G protein G 12 subfamily, has been shown to stimulate c-Jun N-terminal kinase (JNK) activity through the low molecular weight GTP-binding proteins Ras, Rac, and Cdc42. In this study using the transient expression of a constitutively activated mutant of Gα 12 (Gα 12 Q229L) in human embryonic kidney (HEK) 293 cells, we found that Rho and Src family kinase are also involved in the Gα 12-induced activation of JNK. The activation of JNK by Gα 12 Q229L was inhibited by dominant-negative RhoA (T19N), and botulinum C3 exoenzyme which specifically inactivates Rho. In addition, the expression of activated RhoA (G14V) elevated JNK activity in HEK 293 cells. The Gα 12 Q229L-stimulated activation of JNK was blocked by a specific inhibitor of protein tyrosine kinases (PP2), and C-terminal Src kinase (Csk). Moreover, we observed that Gα 12 Q229L stimulated Src family kinase activity and v-Src induced JNK activation. Interestingly, the v-Src-induced activation of JNK was inhibited by dominant-negative RhoA (T19N). In contrast, Csk did not inhibit the JNK activation by activated RhoA (G14V). These results suggest that Rho and Src family kinase are required for the Gα 12-induced JNK activation, and that Src family kinase acts upstream of Rho activation in the JNK pathway.