Rat corticotropin-releasing factor receptor 1 (rCRFR1) was produced either in transfected HEK 293 cells as a complex glycosylated protein or in the presence of the mannosidase I inhibitor kifunensine as a high mannose glycosylated protein. The altered glycosylation did not influence the biological function of rCRFR1 as demonstrated by competitive binding of rat urocortin (rUcn) or human/rat corticotropin-releasing factor (h/rCRF) and agonist-induced cAMP accumulation. The low production rate of the N-terminal domain of rCRFR1 (rCRFR1-NT) by transfected HEK 293 cells, was increased by a factor of 100 in the presence of kifunensine. The product, rCRFR1-NT-Kif, bound rUcn specifically (KD = 27 nM) and astressin (KI = 60 nM). This affinity was 10-fold lower than the affinity of full length rCRFR1. However, it was sufficiently high for rCRFR1-NT-Kif to serve as a model for the N-terminal domain of rCRFR1. With protein fragmentation, Edman degradation, and mass spectrometric analysis, evidence was found for the signal peptide cleavage site C-terminally to Thr23 and three disulfide bridges between precursor residues 30 and 54, 44 and 87, and 68 and 102. Of all putative N-glycosylation sites in positions 32, 38, 45, 78, 90, and 98, all Asn residues except for Asn32 were glycosylated to a significant extent. No O-glycosylation was observed.