To investigate the mechanism for glucocorticoid resistance in leukemic cells, we sequenced the coding region of glucocorticoid receptor (GR) gene in ten Japanese leukemic cells. We identified a novel heterozygous mutation (C643R) in the ligand-binding domain in P30/OHK cells. Western blot analysis for COS-7 cells transfected with the wild-type or C643R mutant GR plasmid revealed similar protein expression levels. In the ligand-binding assay, the dissociation constant of the C643R GR was six-fold higher than that of the wild-type GR. Moreover, the C643R GR showed no transcriptional activity in the luciferase reporter assay.