Endothelin-1 (ET-1) is a vasoconstrictor peptide known to be a potent mitogen for glomerular mesangial cells. We have shown that ET-1 stimulates the adaptor protein p66Shc through Rac/Cdc42 guanine nucleotide exchange factor β 1 Pix. In this study, we demonstrate that ET-1-induced serine phosphorylation of p66Shc is mediated through Gα i3 . Pertussis toxin treatment of cells induced a significant decrease in the interaction between β 1 Pix and ET A -R, and an increase in the binding of Gα i3 and G β1 to β 1 Pix. Activation of heterotrimeric G proteins by AlF 4 − resulted in an increase of Gα i3 binding to β 1 Pix, which was significantly disrupted in cells expressing β 1 Pix dimerization deficient mutant, β 1 PixΔ (602-611). In cells expressing β 1 PixΔ (602-611), ET-1-induced p66Shc activation was also significantly decreased. Specific inhibition of EGF receptor by AG1478 blocked ET-1-induced p66Shc activation and the binding of p66Shc and Gα i3 to β 1 Pix. Inhibition of Erk1/2 blocked p66Shc activation induced by ET-1. Altogether, our results indicate that ET-1 activates p66Shc through EGF receptor transactivation, leading to the activation of Gα i3 , β 1 Pix and Erk1/2.