Agonist-induced phosphorylation of the human corticotropin-releasing factor type 1 receptor (hCRF(1)-R) was investigated using an influenza hemagglutinin (HA) epitope-tagged receptor transiently expressed in COS-7 cells. The HA-hCRF(1)-R migrated as a broad band (M(r) 60,000-70,000) in SDS-PAGE and showed increased mobility (M(r) approximately 48,000) after enzymatic deglycosylation with peptide-N-glycosidase F, consistent with the predicted size (47 kDa) of the nonglycosylated HA-hCRF(1)-R protein. A marked increase in HA-hCRF(1)-R phosphorylation was observed in HA-hCRF(1)-R-expressing COS-7 cells exposed to 1 microM ovine CRF for 5 min, whereas activation of protein kinase A (PKA) by 50 microM forskolin, or of Ca(2+)/calmodulin (CaM)-dependent kinases by 10 microM ionomycin, had little effect. These findings are consistent with preliminary data suggesting that CRF(1)-R phosphorylation mediated by G protein receptor kinase 3 (GRK3), but not by PKA or CaM-dependent kinases, has an important role in the homologous desensitization of brain CRF(1)-Rs.