Activation of the murine c-myc promoter by murine c-Myb protein was examined in several cell lines by using a transient expression system in which Myb expression vectors activate the c-myc promoter linked to a chloramphenicol acetyltransferase reporter gene or a genomic β-globin gene. S1 nuclease protection analyses confirmed that the induction of c-myc by c-Myb was transcriptional and affected both P1 and P2 start sites in a murine T-cell line, EL4, and a myelomonocytic line, WEHI-3. Mutational analyses of the c-myc promoter revealed that two distinct regions could confer Myb responsiveness in two T-cell lines, a distal site upstream of P1 and a proximal site within the first noncoding exon. In contrast, only the proximal site was required for other cell lineages examined. Five separate Myb-binding sites were located in this proximal site and found to be important for c-Myb trans activation. DNA binding was necessary for c-myc activation, as shown by the loss of function associated with mutation of Myb's DNA-binding domain and by trans-dominant repressor activity of the DNA binding, trans-activation-defective mutant. The involvement of additional protein factors was addressed by inhibiting protein synthesis with cycloheximide in a conditional expression system in which the activity of presynthesized Myb was under the control of estrogen. These experiments indicate that de novo synthesis of additional proteins was not necessary for c-myc trans activation. Together these data reveal two cell lineage-dependent pathways by which c-Myb regulates c-myc; however, both pathways are mechanistically indistinguishable in that direct DNA binding by Myb is required for activating c-myc whereas neither de novo protein synthesis nor other labile proteins are necessary.