The alpha isoform of the glucocorticoid receptor (GRalpha) binds glucocorticoids and functions as a ligand-dependent transcription factor. Although GRalpha is expressed in almost all tissues and cells, its subcellular distribution is controversial. Many studies have reported that GRalpha translocates from the cytoplasm to the nucleus in a hormone-dependent manner whereas others have concluded that GRalpha is constitutively located in the nucleus. These conflicting data may result from the use of antibodies that do not discriminate GRalpha from a splice variant of the GR gene termed GRbeta. Using a GRbeta-specific antibody, we have recently demonstrated that GRbeta resides in the nucleus of cells independent of glucocorticoid treatment. In the following study we have generated a novel GRalpha-specific antibody (AShGR) in order to assess, unambiguously, the subcellular distribution of GRalpha. AShGR recognizes recombinant GRalpha on Western blots and in immunoprecipitation experiments but does not cross-react with recombinant GRbeta. Endogenous GRalpha is detected by AShGR in a variety of human cell lines including HeLa S3, CEM-C7, HEK-293, MCF-7, Hep G2, and secondary lung epithelial cells. In addition, AShGR detects endogenous rat and mouse GRalpha. Immunocytochemistry was performed with AShGR on COS-I cells transfected with human GRalpha and on HTC rat hepatoma cells expressing endogenous GRalpha. In both systems, GRalpha was found in the cytoplasm of cells in the absence of hormone and in the nucleus after hormone treatment. These studies mark the first time a GRalpha-specific antibody has been employed to examine the expression and subcellular distribution of endogenous GRalpha.