The GPCR signaling cascade via the G protein pathway begins with an agonist binding to an inactive GPCR, causing conformational changes that activate the GPCR. Previously, we used molecular dynamics simulations to study the activation of the CB2 receptor (a Class A GPCR), by the endogenous ligand, 2-arachidonoylglycerol (2-AG) via the lipid bilayer (Hurst et al., 2010). In the next step of our study of the G-protein signaling cascade, we studied G-protein activation by an activated GPCR. In this step, GDP is released via the separation of the Gαi1 ras-like and helical domains of (Van Eps et al., 2011). To this end, we used our 2-AG activated CB2 model to produce an initial 2-AG/CB2/Gαi1β1γ2 complex based on the crystal structure of β2 adrenoreceptor in complex with Gαsβ1γ2 (Rasmussen et al., 2011). The complex was immersed in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer and NPT NAMD2 (Phillips et al., 2005) molecular dynamics (MD) simulations were initiated for four different trajectories of this system. Results from our longest MD simulation (3μs) suggest that the C-terminal α5 helix of Gαi prefers a different orientation in the CB2 activated receptor relative to the orientation seen in the empty state (GDP-GTP less) β2 adrenoreceptor/Gαsβ1γ2 complex. Initial hydrophobic interactions between P139 on CB2 intracellular loop 2 (IC-2 loop) and a hydrophobic pocket on Gαi consisting of residues V34 (N terminus); F336, T340, I343 and I344 (C terminus); L194 (β1-sheet); and F196 (β2-sheet) stabilize the receptor/Gαi1 protein interface during the first 0.5 μs of the simulation. Later, between 1.4-1.6 μs, electrostatic interactions between R229 (CB2 IC-3 loop) and Q304/E308 (Gαi1 α4 helix) facilitate hydration of GDP.[Support: RO1 DA003934 and KO5 DA021358 (PHR)].