Functional maturation of steroid hormone receptors requires ordered assembly into a large multichaperone complex consisting of receptor monomer, an Hsp90 dimer, the p23 cochaperone, and an FKBP family member or alternate PPIase-related cochaperone. Previous cellular studies demonstrated that FKBP52 can potentiate receptor function. These results have been confirmed in fkbp4 gene knockout mice in which males are partially androgen insensitive and females display characteristics of progesterone insensitivity. Conversely, FKBP51, which has a high degree of similarity to FKBP52, antagonizes FKBP52-mediated potentiation. Both proteins consist of three domains: two FKBP12-like domains termed FK1 and FK2 and a tetratricopeptide repeat domain that targets binding to Hsp90. To help understand why the two FKBPs behave differently and to gain insight into FKBP52 potentiation activity, we have analyzed the loop structure that links FK1 and FK2. Within the FK linker of FKBP52 is the sequence TEEED, which forms a consensus casein kinase II phosphorylation site; the corresponding sequence in FKBP51 is FED. We demonstrate that the distinct FK linker sequences per se do not account for lack of potentiation activity by FKBP51. However, phosphorylation of the FK linker appears to be an important regulatory determinant of FKBP52-mediated potentiation of steroid receptor activity.