Transcriptional enhancement is a general mechanism for regulation of gene expression in which particular proteins bound to specific DNA sequences stimulate the efficiency of initiation from linked promoters. One such protein, the glucocorticoid receptor, mediates enhancement in a glucocorticoid hormone-dependent manner. In this study, a region of the 795-amino acid rat glucocorticoid receptor that is active in transcriptional enhancement was identified. The active region was defined by expressing various receptor deletion mutants in stably and transiently transfected cells and examining the regulated transcription of hormone-responsive genes. Mutant receptors lacking as many as 439 amino-terminal amino acids retained activity, as did those with as many as 270 carboxyl-terminal amino acids deleted. This suggests that the 86-amino acid segment between the most extensive terminal deletions, which also includes sequences required for specific DNA binding in vitro, is sufficient for enhancer activation. In fact, a 150-amino acid receptor fragment that encompasses this segment mediates constitutive enhancement.