The cellular c-myb gene encodes a transcription factor composed of a DNA-binding domain, a transactivating domain and a regulatory domain located at its carboxy (C-) terminus. The latter one is deleted in the transforming viral protein v-Myb. Here we show that deletion of the C-terminus of c-Myb increases the transcriptional transactivation activity of c-Myb defining it as cis-acting negative regulatory domain. Cotransfection of the C-terminus in an in vivo competition assay causes stimulation of the transcriptional activity of various v- and c-Myb expression constructs in trans. The effect is dose-dependent and independent of the kind of DNA-binding domain, since c-Myb as well as GAL4-c-Myb chimaeras can be stimulated in trans. Other transcription factors, such as GAL4-VP16, GAL4, c-Jun or C/EBP beta are also stimulated by the cotransfected C-terminus. In contrast, human B-Myb is not stimulated by the c-Myb C-terminus in trans. The data suggest that the C-terminus of c-Myb may interact with a cellular inhibitor which is part of the protein complex mediating activated transcription and may stimulate in trans by sequestering away such an inhibitor. Binding of c-Myb to a putative inhibitor would explain differences between c-Myb in comparison to B- and v-Myb in transcriptional regulation.