PURPOSE. A spliced variant of the human glucocorticoid receptor GRβ has been implicated in glucocorticoid responsiveness in glaucoma. Over-expression of the FK506-binding immunophilin FKBP51 also causes a generalized state of glucocorticoid resistance. In the present study, the roles of FKBP51 in the nuclear transport of GRβ and glucocorticoid responsiveness were investigated. METHODS. Human trabecular meshwork cells (GTM3 and TM5) and HeLa cells were treated with dexamethasone (DEX) and FK506 and transfected with GRβ and FKBP51 expression vectors. Coimmunoprecipitation and Western blot analyses were performed to study interactions of FKBP51 and FKBP52 with GRa, GRβ, Hsp90, or dynein. The cells were transfected with a GRE-luciferase reporter to evaluate the effects of DEX and FK506 and the overexpression of GRβ and FKBP51 on glucocorticoid-mediated gene expression. RESULTS. FKBP51 was involved in constitutive nuclear transport of both GRa and -β in the absence of ligands. FKBP52 appeared to be solely responsible for the nuclear transport of ligandactivated GRa. DEX stimulated the translocation of GRa but not GRβ. Overexpression of either GRβ or FKBP51 stimulated GRβ translocation and reduced DEX-induced luciferase in HeLa cells. FK506 did not alter DEX-induced translocation of GRa. However, FK506 increased the association of FKBP51 with GRβ and stimulated DEX-induced translocation of GRβ in normal TM cells, but not in glaucoma TM cells. Increased nuclear GRβ significantly inhibited glucocorticoid responsiveness in TM cells. CONCLUSIONS. Nuclear transport of GRβ represents a novel mechanism through which FKBP51 alters GC sensitivity. GRβ and FKBP51 may be responsible for increased responsiveness in steroid-induced ocular hypertensive individuals as well as in patients with glaucoma.