In the present study, we determined the cellular regulators of ERK1/2 and Akt signaling pathways in response to human CRF receptor (CRFR) activation in transfected COS-7 cells. We found that Pertussis Toxin (PTX) treatment or sequestering Gβγ reduced CRFR-mediated activation of ERK1/2, suggesting the involvement of a G-linked cascade. Neither G/PKA nor G/PKC were associated with ERK1/2 activation. Besides, CRF induced EGF receptor (EGFR) phosphorylation at Tyr, and selective inhibition of EGFR kinase activity by AG1478 strongly inhibited the CRFR-mediated phosphorylation of ERK1/2, indicating the participation of EGFR transactivation. Furthermore, CRF-induced ERK1/2 phosphorylation was not altered by pretreatment with batimastat, GM6001, or an HB-EGF antibody indicating that metalloproteinase processing of HB-EGF ligands is not required for the CRF-mediated EGFR transactivation. We also observed that CRF induced Src and PYK2 phosphorylation in a Gβγ-dependent manner. Additionally, using the specific Src kinase inhibitor PP2 and the dominant-negative-SrcYF-KM, it was revealed that CRF-stimulated ERK1/2 phosphorylation depends on Src activation. PP2 also blocked the effect of CRF on Src and EGFR (Tyr) phosphorylation, further demonstrating the centrality of Src. We identified the formation of a protein complex consisting of CRFR, Src, and EGFR facilitates EGFR transactivation and CRFR-mediated signaling. CRF stimulated Akt phosphorylation, which was dependent on G/βγ subunits, and Src activation, however, was only slightly dependent on EGFR transactivation. Moreover, PI3K inhibitors were able to inhibit not only the CRF-induced phosphorylation of Akt, as expected, but also ERK1/2 activation by CRF suggesting a PI3K dependency in the CRFR ERK signaling. Finally, CRF-stimulated ERK1/2 activation was similar in the wild-type CRFR and the phosphorylation-deficient CRFR-Δ386 mutant, which has impaired agonist-dependent β-arrestin-2 recruitment; however, this situation may have resulted from the low β-arrestin expression in the COS-7 cells. When β-arrestin-2 was overexpressed in COS-7 cells, CRF-stimulated ERK1/2 phosphorylation was markedly upregulated. These findings indicate that on the base of a constitutive CRFR/EGFR interaction, the G/βγ subunits upstream activation of Src, PYK2, PI3K, and transactivation of the EGFR are required for CRFR signaling via the ERK1/2-MAP kinase pathway. In contrast, Akt activation via CRFR is mediated by the Src/PI3K pathway with little contribution of EGFR transactivation.