We have used a panel of monoclonal antibodies and a cell-free transcription assay to study the function of the tau 1 transactivation domain of the human glucocorticoid receptor. Three antibodies (monoclonal antibodies 250, 275, and 286) specifically inhibited tau 1-dependent transcription, but had little or no effect on either basal transcription or the activity of an unrelated yeast transcription factor. This inhibition was not due to interference of DNA binding activity, as all three antibodies super shifted tau 1-containing protein-DNA complexes. Epitopes for all three antibodies were localized to a region between amino acids 190 and 200, which lies within the recently defined 41-amino acid core region of tau 1 that is required for transactivation (Dahlman-Wright, K., Almlöf, T., McEwan, I.J., Gustafsson, J-A., and Wright, A. P.H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1619-1623). In contrast to the effect on tau 1-dependent transcription none of the antibodies tested antagonized the squelching ability of the tau 1 domain, suggesting that tau 1-mediated transactivation involves interactions in addition to those identified by the squelching assay. Consistent with this, a comparison of the kinetics of tau 1 squelching and inhibition of transactivation by monoclonal antibodies suggested a role for tau 1 mediated transcriptional induction at two or more steps during transcription initiation by RNA polymerase II.