Papillomaviruses are the causative agents of benign and malignant epithelial tumors of the skin and mucosa. They encode a DNA-binding protein, E2, that regulates viral transcription and replication, making it an important therapeutic target. By deleting the amino-terminal trans-activation domain of human papillomavirus type 16 (HPV-16) E2 while retaining its carboxy-terminal DNA binding and dimerization domain, an E2 repressor (E2R) that efficiently inhibits transcriptional activation by full-length HPV E2 was generated. To deliver this repressor protein into animal cells, we have utilized the human immunodeficiency virus type 1 (HIV-1) Tat protein which itself is taken up efficiently into intact cells. Chimeras of E2R and the cellular uptake domain of Tat specifically inhibited E2-dependent reporter gene expression in COS-7 cells. Treatment of cervical intraepithelial neoplasia cells having episomally replicating HPV-31 DNA with this Tat-E2R protein led to a dose-dependent loss of HPV DNA copies and inhibition of cell growth. Tat-mediated delivery can be a valuable tool for assessing protein function and may allow the development of novel therapeutic proteins having intracellular targets.