The bZIP factor E4BP4 overlaps in DNA binding site specificity with the transcriptional activator CREB and members of the ATF family of transcription factors, but is an active transcriptional repressor. In this study we have mapped the repressing activity of E4BP4 to a small ‘domain’ of 65 amino acids that retains its ability to repress transcription when transferred to the heterologous DNA binding domain of the yeast transcriptional activator GAL4. This segment of the E4BP4 polypeptide contains a high proportion of charged amino acids and does not resemble the repression domains that have been characterized so far from other active transcriptional repressors such as the Drosophila Krüppel, Engralled or Even-skipped proteins. A mutation which changes the charge configuration of this repression module resulted in a complete loss of repressor activity. The E4BP4-GAL4 fusion protein is able to repress the residual transcription from minimal promoters containing the adenovirus E4 or E1 b TATA box. This is consistent with a mechanism of action whereby E4BP4 interacts with some component of the general transcription machinery to cause repression of basal and activated transcription. Although a number of nuclear proteins are able to interact with the E4BP4 repression domain in vitro , these proteins do not appear to include the general transcription factors TFIIB or TBP.