Tyrosine phosphorylation (Tyr-P) of focal adhesion kinase (FAK) regulates FAK activation. Phosphorylated FAK Tyr 397 binds Src family kinases (Src), which in turn directly phosphorylate FAK Tyr 576/577 to produce maximal FAK enzymatic activity. CB cannabinoid receptors (CB) are abundantly expressed in the nervous system and influence FAK activation by presently unknown mechanisms. The current investigation determined that CB-stimulated maximal FAK catalytic activity is mediated by Gi/o proteins in N18TG2 neuronal cells, and that G12/13 regulation of Rac1 and RhoA occurs concomitantly. Immunoblotting analyses using antibodies against FAK phospho-Tyr 397 and phospho-Tyr 576/577 demonstrated that the time-course of CB-stimulated FAK 576/577 Tyr-P occurred in three phases: Phase I (0-2 min) maximal Tyr-P, Phase II (5-20 min) rapid decline in Tyr-P, and Phase III (>20 min) plateau in Tyr-P at submaximal levels. In contrast, FAK 397 Tyr-P was monophasic and significantly lower in magnitude. FAK 397 Tyr-P and Phase I FAK 576/577 Tyr-P involved protein tyrosine phosphatase (PTP1B and Shp1/Shp2)-mediated Src activation, Protein Kinase A (PKA) inhibition, and integrin activation. Phase I maximal FAK 576/577 Tyr-P also required cooperative signaling between receptor tyrosine kinases (RTKs) and integrins. The integrin antagonist RGDS peptide, Flk-1 vascular endothelial growth factor receptor (VEGFR) antagonist SU5416, and epidermal growth factor receptor (EGFR) antagonist AG 1478 blocked Phase I FAK 576/577 Tyr-P. CB agonists failed to stimulate FAK Tyr-P in the absence of integrin activation upon suspension in serum-free culture media. In contrast, cells grown on the integrin ligands fibronectin and laminin displayed increased FAK 576/577 Tyr-P that was augmented by CB agonists and blocked by the Src inhibitor PP2 and Flk-1 VEGFR antagonist SU5416. Taken together, these studies have identified a complex integrative pathway utilized by CB to stimulate maximal FAK 576/577 Tyr-P in neuronal cells.