The type 1 corticotropin-releasing hormone receptor (CRHR1) influences biological responses important for adaptation to stressful stimuli, through activation of multiple downstream effectors. The structural motifs within CRH-R1 that mediate G protein activation and signaling selectivity are unknown. The aim of this study was to gain insights about important structural determinants within the third intracellular loop (IC3) of the human CRH-R1α important for cAMP and ERK1/2 pathways activation and selectivity. We investigated the role of the juxtamembrane regions of IC3 by mutating amino acid cassettes or specific residues to alanine. Although simultaneous tandem alanine mutations of both juxtamembrane regions Arg 292-Met 295 and Lys 311-Lys 314 reduced ligand binding and impaired signaling, all other mutant receptors retained high affinity binding, indistinguishable from wild-type receptor. Agonist-activated receptors with tandem mutations at the proximal or distal terminal segments enhanced activation of adenylyl cyclase by 50-75%and diminished activation of inositol trisphosphate and ERK1/2 by 60-80%. Single Ala mutations identified Arg 292, Lys 297, Arg 310, Lys 311, and Lys 314 as important residues for the enhanced activation of adenylyl cyclase, partly due to reduced inhibition of adenylyl cyclase activity by pertussis toxin-sensitive G proteins. In contrast, mutation of Arg 299 reduced receptor signaling activity and cAMP response. Basic as well as aliphatic amino acids within both juxtamembrane regions were identified as important for ERK1/2 phosphorylation through activation of pertussis toxin-sensitive G proteins as well as G q proteins. These data uncovered unexpected roles for key amino acids within the highly conserved hydrophobic N- and C-terminal microdomains of IC3 in the coordination of CRH-R1 signaling activity.