CCRF-CEM-C7 is a well characterized human leukemic clonal cell line which is lysed by dexamethasone (dex). Originating from the wild-type CEM-C7 cells are two dex-resistant clones which are not lysed by 1 microM dex and have functionally defective glucocorticoid receptors (GR). They are receptorless ICR27TK.3 and activation-labile 4R4 cells. ICR27TK.3 and 4R4 cells have distinct cellular phenotypes, as indicated by dissimilar numbers of dex-binding sites despite similar levels of GR mRNA and immunochemically detectable GR. We have now investigated the molecular defects in the GR of ICR27TK.3 and 4R4 cells by determining the nucleotide sequence of their GR. Our results support the biochemical evidence previously reported by others for the presence of both a normal (GR+) and a mutant (GR*) allele in CEM-C7 cells. We clearly show that the wild-type CEM-C7 cells express two alleles of GR, the normal GR+ and the abnormal GR*, which has a Leu753--> Phe753 mutation. We demonstrate that both ICR27TK.3 and 4R4 cells contain only the abnormal GR* and that the normal GR+ gene is deleted in both of these GR defective clones. Our results further show that the GR* is basically an activation-labile receptor and has diminished functional capability in a transfection assay measuring GR-driven transcription. Thus, these two phenotypically different cell lines express similar amounts of an identical GR* containing a single point mutation at amino acid 753. A single point mutation in the steroid-binding domain of the GR, therefore, may behave differently, depending on the cellular milieu in which it is expressed.