Although thiazolidinediones were designed as specific peroxisome proliferator-activated receptor (PPAR)-γ-ligands, there is evidence for some off-target effects mediated by a non-PPARγ mechanism. Previously we have shown that rosiglitazone has antiinflammatory actions not explicable by activation of PPARγ,but possibly by the glucocorticoid receptor (GR). Rosiglitazone induces nuclear translocation both of GR-green fluorescent protein, and endogenous GR in HeLa and U20S cells but with slower kinetics than dexamethasone. Rosiglitazone also induces GR phosphorylation (Ser211), a GR ligand-binding-specific effect. Rosiglitazone drives luciferase expression from a simple glucocorticoid-response element containing reporter gene in a GR-dependent manner (EC50 4 μm), with a similar amplitude response to the partial GR agonist RU486. Rosiglitazone also inhibits dexamethasone-driven reporter gene activity (IC50 2.9 μm) in a similar fashion to RU486, suggesting partial agonist activity. Importantly we demonstrate a similar effect in PPARγ-null cells, suggesting both GR dependence and PPARγ independence. Rosiglitazone also activates a GAL4-GR chimera, driving a upstream activating sequence promoter, demonstrating DNA template sequence independence and furthermore enhanced steroid receptor coactivator-1-GR interaction, measured by a mammalian two-hybrid assay. Both ciglitazone and pioglitazone, structurally related to rosiglitazone, show similar effects on the GR. The antiproliferative effect of rosiglitazone is increased in U20S cells that overexpress GR, suggesting a biologically important GR-dependent component of rosiglitazone action. Rosiglitazone is a partial GR agonist, affecting GR activation and trafficking to influence engagement of target genes and affect cell function. This novel mode of action may explain some off-target effects observed in vivo. Additionally, antagonism of glucocorticoid action may contribute to the antidiabetic actions of rosiglitazone. Rosiglitazone, ciglitazone, and pioglitazone are partial glucocorticoid receptor (GR) agonists, affecting GR phosphorylation, subcellular trafficking, co-modulator recruitment, and transcription regulatory function independently of peroxisome proliferator-activated receptorã.