The c-Myb protein binds to DNA, can regulate transcription, and is required for normal hematopoiesis in vertebrates. Either amino- or carboxy-terminal truncation of this protein is required for efficient oncogenic activation. Previous studies have shown that the carboxyl terminus of c-Myb that is deleted in v-Myb contains negative regulatory domains. We now demonstrate that specific mutations within this carboxyl terminus result in greater transcriptional activation than truncation of the entire carboxyl terminus. Furthermore, this increased transcriptional activation depends upon the presence of the highly conserved Myb DNA-binding domain and is also dependent upon the nature of the Myb-binding sites within the target promoter. In a similar fashion, an activating mutation within the heptad leucine repeat region of c-Myb that is also present in v-Myb functions only in conjunction with the Myb DNA-binding domain and with particular Myb-binding sites. These results suggest a model in which multiple domains of the c-Myb protein are highly interdependent for transcriptional regulation. These interactions are promoter-specific and are not well modeled by heterologous fusion proteins.