The trans-activating and transforming capacities of the c-myb proto-oncogene product (c-Myb) are negatively regulated through a leucine zipper structure in its negative regulatory domain. We show here tht in cotransfection assays, maximal Myb-induced trans-activation occurs with relatively low amounts of wild-type c-Myb, while higher levels of c-Myb result in reduced Myb-induced trans-activation. By contrast, this apparent negative autoregulation is not observed with a c-Myb mutant containing an impaired leucine zipper. Data presented here suggest that this negative autoregulation of trans-activation by wild-type c-Myb is a consequence of homodimer formation by c-Myb through its leucine zipper and of the inability of c-Myb dimers to bind DNA. These findings point to a novel mechanism of regulation of a transcription factor.