The effects of steroid hormones are mediated by intracellular hormone-specific receptor proteins; the interaction between the hormone and its receptor increases the affinity of the receptor for nuclear binding sites, thereby modulating the expression of specific genes. The glucocorticoid receptor is a soluble protein of relative molecular mass (Mr) 94,000 (94K), present at a low relative abundance (less than or equal to 0.01%); it has been purified to near-homogeneity, and specific antisera and monoclonal antibodies have been produced. Purified glucocorticoid receptor binds in vitro with high affinity to defined regions of DNA near regulated promoters, and sequences essential for these interactions are functional in vivo as hormone-dependent transcriptional enhancer elements. We have now cloned complementary DNA (cDNA) for the rat liver glucocorticoid receptor and we describe here a 2.6-kilobase (kb) receptor cDNA isolated following polysome immuno-enrichment of receptor messenger RNA with glucocorticoid receptor-specific antibodies. The receptor appears to be encoded by a single-copy gene which specifies a approximately 6-kb transcript in rat and mouse cells; this mRNA is altered quantitatively and qualitatively in several mutant cell lines with specific defects in receptor function.