The corticotropin-releasing factor receptor 1 (CRFR1) and serotonin 2A receptor (5-HT2AR) are linked to cellular mechanisms underlying stress anxiety and depression. Both receptors are members of the G protein-coupled receptor (GPCR) superfamily and encode class I PSD-95/DiscsLarge/Zona Occludens 1 (PDZ) binding motifs (-S/T-x-V/I/L) at the end of their carboxyl-terminal tails. We have identified PDZK1, also referred to as Na(+)/H(+) exchange regulatory cofactor 3 (NHERF3) as both a CRFR1- and 5-HT2AR-interacting protein. We have examined whether PDZK1 plays a role in regulating both CRFR1 and 5-HT2AR activity. We find that while PDZK1 interactions with CRFR1 are PDZ binding motif-dependent, PDZK1 associates with 5-HT2AR in a PDZ binding motif-independent manner and CRFR1 expression, but not 5-HT2AR expression, redistributes PDZK1 to the plasma membrane in PDZ binding motif-dependent manner. PDZK1, negatively regulates 5-HT2AR endocytosis and has no effect upon 5-HT2AR-mediated ERK1/2 phosphorylation. In contrast, PDZK1 overexpression does not affect CRFR1 endocytosis, but selectively increases CRFR1-stimulated ERK1/2 phosphorylation. Similar to what has been previously reported for PSD-95 and SAP97, PDZK1 positively influences 5-HT2AR-stimulated inositol phosphate formation, but does not contribute to the regulation of CRFR1-mediated cAMP signaling. Taken together, these results indicate that PDZK1 differentially regulates the signaling and trafficking of CRFR1 and 5-HT2AR via PDZ-dependent and -independent mechanisms, respectively.