The hormone-binding competent glucocorticoid receptor (GR) is found in complex with a heat shock-binding protein 90 (Hsp90) dimer, p23 and one member of a group of cochaperones termed immunophilins. Selective incorporation of the immunophilins FKBP52 or FKBP51 into the mature GR heterocomplex results in an upregulation or inhibition of receptor action, respectively. The mechanism by which immunophilins act upon the GR ligand-binding domain (LBD) is unknown. Decreased cortisol sensitivity in the guinea pig has been attributed to five residue changes within the helix 1 to helix 3 (H1-H3) loop of the guinea pig GR LBD. It has been hypothesised that this loop might serve as a contact point for FKBP52 and/or FKBP51 with the receptor. Using mouse embryo fibroblast (MEF) knockout models, a comparison of hormone-dependent transcriptional activity between human GR and a receptor containing the guinea pig GR mutations was performed. Our study revealed that loss of FKBP52 negated the differential hormone sensitivity between human and guinea pig GR suggesting that the amino acid changes in the H1-H3 loop favour an interaction with FKBP51 over FKBP52. This was further demonstrated in a transcriptional assay in MEF FKBP51 knockout cells where FKBP51 overexpression resulted in significant attenuation of receptor activity in GR containing the guinea pig mutations. We conclude that the loop provides an additional modulatory surface for GR regulation by the FKBP cochaperones. The H1-H3 loop may impact allosterically on proximally located LBD residues that contribute to GR Hsp90-dependent stability, nuclear translocation and hormone-induced conformational changes, features that are crucial for immunophilin-mediated modulation of GR.