A glucocorticoid receptor(GR) interacting protein JAB1 was isolated from human marrow cDNA library by two-hybrid screening in yeast using the GR ligand-binding domains (GR-LBD) as bait. To further demonstrate the interaction between GR and JAB1 and the effect of JAB1 on GR, PCR was performed to amplify GR-LBD fragments and it was cloned into the bait vector pGBKT7 to create the plasmid pGBKT7-GR LBD. The plasmid was used as bait to screen a cDNA library constructed in the pACT2 vector. Transformants were plated on SD/－Ade/－His/－Leu/－Trp. Yeast colonies were assayed for α-galactosidase activity. The positive colonies were sequenced in which JAB1 cDNA fragments were cloned into the vector pGEX-4T-1 for GST pull analysis. A GRE-driven reporter gene was used for CAT activity assay. The results were as follows: 42 clones turned blue in the yeast α-galactosidase assay and contained pACT2 sequence in which 5 clones corresponded to a fragment of JAB1 as shown by DNA sequencing. Yeast two-hybrid and GST pull down assay verified that JAB1 interacted with GR-LBD. GR, a GRE-driven reporter gene cotransfected with JAB1, strongly potentiated the transactivation properties of GR. The studies suggest that JAB1 interacts with GR-LBD expressed in COS7 cells in vitro and it potentiates the transactivation properties of GR.